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Sample records for akt phosphorylation induced

  1. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

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    Yang, Lifen [Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan (China); Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, IL 60637 (United States); The Committees on Immunology, The University of Chicago, Chicago, IL 60637 (United States); Qiao, Guilin; Ying, Haiyan [Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, IL 60637 (United States); The Committees on Immunology, The University of Chicago, Chicago, IL 60637 (United States); Zhang, Jian, E-mail: jzhang@medicine.bsd.uchicago.edu [Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan (China); Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, IL 60637 (United States); The Committees on Immunology, The University of Chicago, Chicago, IL 60637 (United States); The Committees on Molecular Medicine, The University of Chicago, Chicago, IL 60637 (United States); Yin, Fei, E-mail: yf2323@hotmail.com [Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan (China)

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

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

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

    2001-01-01

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

  3. Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus

    International Nuclear Information System (INIS)

    Expression of the JSRV envelope (Env) is sufficient to transform immortalized rodent fibroblasts. A putative docking site for the PI3-K kinase (Y590-X-X-M593) in the cytoplasmic tail of the transmembrane domain of the JSRV Env is a major determinant of viral-induced cell transformation. Akt is constitutively phosphorylated in rodent fibroblasts transformed by the JSRV Env. However, recent data suggest that Y590 and M593 are not necessary for JSRV Env-induced transformation of the immortalized chicken fibroblasts cell line DF-1. In this study we found that JSRV-induced transformation of DF-1 cells is Akt-independent. In addition, a replication-competent avian vector expressing the JSRV Env (RCASBP(A)+JE) was also able to induce transformation of primary chicken embryo fibroblasts (CEF). Vectors expressing JSRV Env Y590 mutants were still able to induce CEF cells transformation but not as efficiently as the vectors expressing the wild-type Env. In CEF cells, as in DF-1 cells, only the expression of the wild-type Env induced constitutive phosphorylation of Akt. Thus, in chicken cells, the degree of transformation induced by the JSRV Env is maximum in the presence of Y590 and Akt phosphorylation. We addressed the significance of Akt phosphorylation in rat 208F cells transformed by the JSRV Env and showed that Akt is indeed activated and shows kinase activity. Inhibitors of the PI-3K/Akt pathway reproducibly decreased the transformation efficiency of the JSRV Env. In vivo, we found phosphorylated Akt only in nasal tumors induced by the enzootic nasal tumor virus (ENTV), a JSRV-related β-retrovirus. No evidence of Akt phosphorylation was found in lung tumor sections of sheep affected by pulmonary adenocarcinoma. As a whole, these results suggest that the activation of the PI-3K/Akt pathway contributes to the process of JSRV-induced cell transformation but most likely is not the primary determinant both in vitro and in vivo

  4. AKT inhibitor suppresses hyperthermia-induced Ndrg2 phosphorylation in gastric cancer cells

    International Nuclear Information System (INIS)

    Hyperthermia is one of the most effective adjuvant treatments for various cancers with few side effects. However, the underlying molecular mechanisms still are not known. N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor, has been shown to be involved in diverse cellular stresses including hypoxia, lipotoxicity, etc. In addition, Ndrg2 has been reported to be related to progression of gastric cancer. In the current study, our data showed that the apoptosis rate of MKN28 cells increased relatively rapidly to 13.4% by 24 h after treatment with hyperthermia (42°C for 1 h) compared to 5.1% in control cells (P < 0.05). Nevertheless, there was no obvious change in the expression level of total Ndrg2 during this process. Further investigation demonstrated that the relative phosphorylation levels of Ndrg2 at Ser332, Thr348 increased up to 3.2- and 1.9-fold (hyperthermia group vs control group) at 3 h in MKN28 cells, respectively (P < 0.05). We also found that heat treatment significantly increased AKT phosphorylation. AKT inhibitor VIII (10 µM) decreased the phosphorylation level of Ndrg2 induced by hyperthermia. Accordingly, the apoptosis rate rose significantly in MKN28 cells (16.4%) treated with a combination of AKT inhibitor VIII and hyperthermia compared to that (6.8%) of cells treated with hyperthermia alone (P < 0.05). Taken together, these data demonstrated that Ndrg2 phosphorylation could be induced by hyperthermia in an AKT-dependent manner in gastric cancer cells. Furthermore, AKT inhibitor VIII suppressed Ndrg2 phosphorylation and rendered gastric cancer cells susceptible to apoptosis induced by hyperthermia

  5. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

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    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates); Galadari, Sehamuddin, E-mail: sehamuddin@uaeu.ac.ae [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  6. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    International Nuclear Information System (INIS)

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  7. mTORC2 phosphorylation of Akt1: a possible mechanism for hydrogen sulfide-induced cardioprotection.

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    Yue Zhou

    Full Text Available Hydrogen sulfide (H2S is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i normoxia, (ii global ischemia and (iii ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3 vs. 10±3×10(3 mmHg/min, p<0.05 and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05 compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05. Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism

  8. Human recombinant H2 relaxin induces AKT and GSK3β phosphorylation and HTR-8/SVneo cell proliferation.

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    Astuti, Yoni; Nakabayashi, Koji; Deguchi, Masashi; Ebina, Yasuhiko; Yamada, Hideto

    2015-01-01

    Relaxin is essential for trophoblast development during pregnancy. Evidence shows that relaxin increases trophoblast cell migration capacity. Here, we show the effect of relaxin on protein kinase B (AKT) activation and glycogen synthase kinase 3-beta (GSK3β) inactivation as well as on the proliferation of HTR-8/SVneo cells, a model of human extravillous trophoblast (EVT). HTR-8/SVneo cells were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions and treated for increasing time with 1 ng/mL of rH2 relaxin. Western blot analysis was performed to detect pAKT, AKT, pGSK3β, GSK3β, and actin expression. Proliferation of HTR-8/SVneo cells was analyzed by MTS assay. rH2 relaxin treatment increased the ratio of pAKT/AKT, pGSK3β/GSK3β, and proliferation in HTR-8/SVneo cells. Furthermore, AKT and GSK3β activation by rH2 relaxin was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor. This study suggests that rH2 relaxin induces AKT and GSK3β phosphorylation as well as proliferation in HTR-8/SVneo cells. PMID:25868609

  9. Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D

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    Razmara Masoud

    2013-01-01

    Full Text Available Abstract Mammalian target of rapamycin (mTOR can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor and mTORC2 (containing Rictor. Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1 and protein kinase C (PKC was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD. It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner

  10. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

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    Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  11. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    International Nuclear Information System (INIS)

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  12. The HIV Protease Inhibitor Nelfinavir Downregulates Akt Phosphorylation by Inhibiting Proteasomal Activity and Inducing the Unfolded Protein Response

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    Anjali K. Gupta

    2007-04-01

    Full Text Available HIV protease inhibitors (HPIs, which have been used to treat HIV patients since the mid 1990s, have been shown to downregulate the phosphatidylinositol 3kinase (PI3K-Akt pathway. Because this pathway is frequently activated in human malignancies and associated with resistance to ionizing radiation, we investigated and confirmed that HPIs could radiosensitize cells. However, the mechanism underlying this downregulation was unclear, prompting the investigations in this report. In this paper we show that nelfinavir inhibits proteasome activity. Inhibition of the proteasome leads to endoplasmic reticulum-based stress with accumulation of misfolded proteins, which triggers the unfolded protein response (UPR. As part of the UPR, the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α is phosphorylated, resulting in a decrease in global protein synthesis and induction of the feedback regulator growth arrest and DNA damageinducible protein (GADD34, which acts as a phosphatase in complex with protein phosphatase 1. This complex dephosphorylates eIF2α; however, our data also suggest that this phosphatase activity can dephosphorylate Akt. Furthermore, our data indicate that nelfinavir decreases Akt phosphorylation by triggering this response. These findings may have important implications in understanding how nelfinavir may increase radiation sensitivity and also result in downregulation of the PI3K/Akt pathway.

  13. Mouse hippocampal phosphorylation footprint induced by generalized seizures: Focus on ERK, mTORC1 and Akt/GSK-3 pathways.

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    Gangarossa, Giuseppe; Sakkaki, Sophie; Lory, Philippe; Valjent, Emmanuel

    2015-12-17

    Exacerbated hippocampal activity has been associated to critical modifications of the intracellular signaling pathways. We have investigated rapid hippocampal adaptive responses induced by maximal electroshock seizure (MES). Here, we demonstrate that abnormal and exacerbated hippocampal activity induced by MES triggers specific and temporally distinct patterns of phosphorylation of extracellular signal-related kinase (ERK), mammalian target of rapamycin complex (mTORC) and Akt/glycogen synthase kinase-3 (Akt/GSK-3) pathways in the mouse hippocampus. While the ERK pathway is transiently activated, the mTORC1 cascade follows a rapid inhibition followed by a transient activation. This rebound of mTORC1 activity leads to the selective phosphorylation of p70S6K, which is accompanied by an enhanced phosphorylation of the ribosomal subunit S6. In contrast, the Akt/GSK-3 pathway is weakly altered. Finally, MES triggers a rapid upregulation of several plasticity-associated genes as a consequence exacerbated hippocampal activity. The results reported in the present study are reminiscent of the one observed in other models of generalized seizures, thus defining a common molecular footprint induced by intense and aberrant hippocampal activities. PMID:26545981

  14. Insulin-like growth factor-1 (IGF-1 induces the activation/phosphorylation of Akt kinase and cAMP response element-binding protein (CREB by activating different signaling pathways in PC12 cells

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    Zheng Wen-Hua

    2006-06-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is a polypeptide growth factor with a variety of functions in both neuronal and non-neuronal cells. IGF-1 plays anti-apoptotic and other functions by activating multiple signaling pathways including Akt kinase, a serine/threonine kinase essential for cell survival. The nuclear transcription factor cAMP response element-binding protein (CREB may also be involved although relationships between these two proteins in IGF-1 receptor signaling and protection is not clear, especially in neuronal cells. Results IGF-1, in a concentration- and time-dependent manner, induces the activation/phosphorylation of Akt and CREB in PC12 cells by activating different signaling pathways. IGF-1 induced a sustained phosphorylation of Akt while only a transient one was seen for CREB. The phosphorylation of Akt is mediated by the PI3 kinase pathway while that of CREB is dependent on the activation of both MAPK kinase and p38 MAPK. Moreover, the stimulation of PKC attenuated the phosphorylation of Akt induced by IGF-1 while enhancing that of CREB. Survival assays with various kinase inhibitors suggested that the activation/phosphorylation of both Akt and CREB contributes to IGF-1 mediated cell survival in PC12 cells. Conclusion These data suggest that IGF-1 induced the activation of Akt and CREB using distinct pathways in PC12 cells.

  15. A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy

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    Watanabe Hirotsuna

    2009-11-01

    Full Text Available Abstract Background The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies. Results Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not. Conclusion Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

  16. FV-429 induces apoptosis and inhibits glycolysis by inhibiting Akt-mediated phosphorylation of hexokinase II in MDA-MB-231 cells.

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    Zhou, Yuxin; Lu, Na; Qiao, Chen; Ni, Ting; Li, Zhiyu; Yu, Boyang; Guo, Qinglong; Wei, Libin

    2016-09-01

    In this study, the anticancer effect of a newly synthesized flavonoid FV-429, against human breast cancer MDA-MB-231 cells, and the underlying mechanisms were investigated. FV-429 triggered the apoptosis and simultaneously inhibited the glycolysis of MDA-MB-231 cells. Both the HK II activity and its level in mitochondria were significantly down regulated by FV-429. Moreover, FV-429 weakened the interaction between HKII and VDAC, stimulated the detachment of HK II from the mitochondria, and resulted in the opening of the mitochondrial permeability transition pores. Thus FV-429 induced the mitochondrial-mediated apoptosis, showing increased Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (MMP) and activation of caspase-3 and -9, cytochrome c (Cyt c) release, and apoptosis inducing factor (AIF) transposition. Further research revealed that the phosphorylation of mitochondrial HKII via Akt was responsible for the dissociation of HKII and the decreased HKII activity induced by FV-429. Taken together, FV-429 inhibited the phosphorylation of HKII, down-regulated its activity, and stimulated the release of HKII from the mitochondria, resulting the inhibited glycolysis and mitochondrial-mediated apoptosis. The studies provide a molecular basis for the development of flavonoid compounds as novel anticancer agents for breast cancer. © 2015 Wiley Periodicals, Inc. PMID:26258875

  17. Phosphorylation and inactivation of PTEN at residues Ser380/Thr382/383 induced by Helicobacter pylori promotes gastric epithelial cell survival through PI3K/Akt pathway.

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    Yang, Zhen; Xie, Chuan; Xu, Wenting; Liu, Gongmeizi; Cao, Ximei; Li, Wei; Chen, Jiang; Zhu, Yin; Luo, Shiwen; Luo, Zhijun; Lu, Nonghua

    2015-10-13

    Phosphorylation of PTEN at residues Ser380/Thr382/383 leads to loss of phosphatase activity and tumor suppressor function. Here, we found that phosphorylation of PTEN at residues Ser380/Thr382/383 was increased with gastric carcinogenesis, and more importantly, Helicobacter pylori was a trigger of this modification in chronic non-atrophic gastritis. H. pylori could phosphorylate and inactivate PTEN in vivo and in vitro, resulting in survival of gastric epithelial cells. Furthermore, stable expression of dominant-negative mutant PTEN or inhibition of Akt prevented the enhanced survival induced by H. pylori. These results indicate that PTEN phosphorylation at residues Ser380/Thr382/383 is a novel mechanism of PTEN inactivation in gastric carcinogenesis, and H. pylori triggers this modification, resulting in activation of the PI3K/Akt pathway and promotion of cell survival. PMID:26376616

  18. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

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    Ilowski, Maren; Putz, Christine [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Weiss, Thomas S. [Department of Surgery, University of Regensburg Hospital, Regensburg (Germany); Brand, Stephan [Department of Internal Medicine II, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, TU Dortmund University, Dortmund (Germany); Thasler, Wolfgang Erwin, E-mail: wolfgang.thasler@med.uni-muenchen.de [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany)

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  19. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

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    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

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

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    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2012-08-17

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

  1. Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival.

    Science.gov (United States)

    Costa, Adriana Fraga; Gomes, Sara Zago; Lorenzon-Ojea, Aline R; Martucci, Mariane; Faria, Miriam Rubio; Pinto, Décio Dos Santos; Oliveira, Sergio F; Ietta, Francesca; Paulesu, Luana; Bevilacqua, Estela

    2016-05-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation. PMID:27208405

  2. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Takehara, Toshiyuki; Onodera, Yuta [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Nakagawa, Koichi; Hamanishi, Chiaki [Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan); Fukuda, Kanji [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nijkamp, Monique M.; Span, Paul N.; Stegeman, Hanneke [Department of Radiation Oncology, Radboud University Nijmegen Medical Center, Nijmegen (Netherlands); Grénman, Reidar [Departments of Otorhinolaryngology-Head-and-Neck-Surgery and Medical Biochemistry, Turku University Hospital and University of Turku, Turku (Finland); Kaanders, Johannes H.A.M. [Department of Radiation Oncology, Radboud University Nijmegen Medical Center, Nijmegen (Netherlands); Bussink, Johan, E-mail: j.bussink@rther.umcn.nl [Department of Radiation Oncology, Radboud University Nijmegen Medical Center, Nijmegen (Netherlands)

    2013-10-01

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

  4. Diaminothiazoles inhibit angiogenesis efficiently by suppressing Akt phosphorylation.

    Science.gov (United States)

    Thomas, Sannu A; Thamkachy, Reshma; Ashokan, Bindu; Komalam, Reena J; Sreerekha, Keerthi V; Bharathan, Asha; Santhoshkumar, Thankayyan R; Rajasekharan, Kallikat N; Sengupta, Suparna

    2012-06-01

    The prevention of neovessel formation or angiogenesis is a recent popular strategy for limiting and curing cancer. Diaminothiazoles are a class of compounds that have been reported to show promise in the treatment of cancer by inhibiting cancer cell proliferation and inducing apoptosis, because of their effects on microtubules and as inhibitors of cyclin-dependent kinases. Many microtubule-targeting agents are being studied for their antiangiogenic activity, and a few have shown promising activity in the treatment of cancer. Here, we report that diaminothiazoles can be highly effective as antiangiogenic agents, as observed in the chick membrane assay. The lead compound, 4-amino-5-benzoyl-2-(4-methoxyphenylamino)thiazole (DAT1), inhibits endothelial cell processes such as invasion, migration, and tubule formation, which require a functional cytoskeleton. DAT1 also decreases the expression of cell adhesion markers. The antiangiogenic activities of DAT1 occur at concentrations that are not cytotoxic to the normal endothelium. Analysis of intracellular signaling pathways shows that DAT1 inhibits Akt phosphorylation, which is actively involved in the angiogenic process. The antiangiogenic properties of diaminothiazoles, in addition to their promising antimitotic and cytotoxic properties in cancer cell lines, give them an extra advantage in the treatment of cancer. PMID:22414853

  5. DSePA Antagonizes High Glucose-Induced Neurotoxicity: Evidences for DNA Damage-Mediated p53 Phosphorylation and MAPKs and AKT Pathways.

    Science.gov (United States)

    Wang, Kun; Fu, Xiao-Yan; Fu, Xiao-Ting; Hou, Ya-Jun; Fang, Jie; Zhang, Shuai; Yang, Ming-Feng; Li, Da-Wei; Mao, Lei-Lei; Sun, Jing-Yi; Yuan, Hui; Yang, Xiao-Yi; Fan, Cun-Dong; Zhang, Zong-Yong; Sun, Bao-Liang

    2016-09-01

    Hyperglycemia as the major hallmark of diabetic neuropathy severely limited its therapeutic efficiency. Evidences have revealed that selenium (Se) as an essential trace element could effectively reduce the risk of neurological diseases. In the present study, 3,3'-diselenodipropionic acid (DSePA), a derivative of selenocystine, was employed to investigate its protective effect against high glucose-induced neurotoxicity in PC12 cells and evaluate the underlying mechanism. The results suggested that high glucose showed significant cytotoxicity through launching mitochondria-mediated apoptosis in PC12 cells, accompanied by poly (ADP-ribose) polymerase (PARP) cleavage, caspase activation, and mitochondrial dysfunction. Moreover, high glucose also triggered DNA damage and dysregulation of MAPKs and AKT pathways through reactive oxygen species (ROS) overproduction. p53 RNA interference partially suppressed high glucose-induced cytotoxicity and apoptosis, indicating the role of p53 in high glucose-induced signal. However, DSePA pretreatment effectively attenuated high glucose-induced cytotoxicity, inhibited the mitochondrial dysfunction through regulation of Bcl-2 family, and ultimately reversed high glucose-induced apoptotic cell death in PC12 cells. Attenuation of caspase activation, PARP cleavage, DNA damage, and ROS accumulation all confirmed its protective effects. Moreover, DSePA markedly alleviated the dysregulation of AKT and MAPKs pathways induced by high glucose. Our findings revealed that the strategy of using DSePA to antagonize high glucose-induced neurotoxicity may be a highly effective strategy in combating high glucose-mediated neurological diseases. PMID:26232068

  6. Cellular stress-induced up-regulation of FMRP promotes cell survival by modulating PI3K-Akt phosphorylation cascades

    Directory of Open Access Journals (Sweden)

    Wells David

    2011-02-01

    Full Text Available Abstract Background Fragile X syndrome (FXS, the most commonly inherited mental retardation and single gene cause of autistic spectrum disorder, occurs when the Fmr1 gene is mutated. The product of Fmr1, fragile X linked mental retardation protein (FMRP is widely expressed in HeLa cells, however the roles of FMRP within HeLa cells were not elucidated, yet. Interacting with a diverse range of mRNAs related to cellular survival regulatory signals, understanding the functions of FMRP in cellular context would provide better insights into the role of this interesting protein in FXS. Using HeLa cells treated with etoposide as a model, we tried to determine whether FMRP could play a role in cell survival. Methods Apoptotic cell death was induced by etoposide treatment on Hela cells. After we transiently modulated FMRP expression (silencing or enhancing by using molecular biotechnological methods such as small hairpin RNA virus-induced knock down and overexpression using transfection with FMRP expression vectors, cellular viability was measured using propidium iodide staining, TUNEL staining, and FACS analysis along with the level of activation of PI3K-Akt pathway by Western blot. Expression level of FMRP and apoptotic regulator BcL-xL was analyzed by Western blot, RT-PCR and immunocytochemistry. Results An increased FMRP expression was measured in etoposide-treated HeLa cells, which was induced by PI3K-Akt activation. Without FMRP expression, cellular defence mechanism via PI3K-Akt-Bcl-xL was weakened and resulted in an augmented cell death by etoposide. In addition, FMRP over-expression lead to the activation of PI3K-Akt signalling pathway as well as increased FMRP and BcL-xL expression, which culminates with the increased cell survival in etoposide-treated HeLa cells. Conclusions Taken together, these results suggest that FMRP expression is an essential part of cellular survival mechanisms through the modulation of PI3K, Akt, and Bcl-xL signal

  7. Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition

    Science.gov (United States)

    Katayama, Kazuhiro; Fujita, Naoya; Tsuruo, Takashi

    2005-01-01

    The serine/threonine kinase Akt is known to promote cell growth by regulating the cell cycle in G1 phase through activation of cyclin/Cdk kinases and inactivation of Cdk inhibitors. However, how the G2/M phase is regulated by Akt remains unclear. Here, we show that Akt counteracts the function of WEE1Hu. Inactivation of Akt by chemotherapeutic drugs or the phosphatidylinositide-3-OH kinase inhibitor LY294002 induced G2/M arrest together with the inhibitory phosphorylation of Cdc2. Because the increased Cdc2 phosphorylation was completely suppressed by wee1hu gene silencing, WEE1Hu was associated with G2/M arrest induced by Akt inactivation. Further analyses revealed that Akt directly bound to and phosphorylated WEE1Hu during the S to G2 phase. Serine-642 was identified as an Akt-dependent phosphorylation site. WEE1Hu kinase activity was not affected by serine-642 phosphorylation. We revealed that serine-642 phosphorylation promoted cytoplasmic localization of WEE1Hu. The nuclear-to-cytoplasmic translocation was mediated by phosphorylation-dependent WEE1Hu binding to 14-3-3θ but not 14-3-3β or -σ. These results indicate that Akt promotes G2/M cell cycle progression by inducing phosphorylation-dependent 14-3-3θ binding and cytoplasmic localization of WEE1Hu. PMID:15964826

  8. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    International Nuclear Information System (INIS)

    Highlights: → PARP1 inhibitors cause a cytotoxic effect independent of DNA repair impairment. → PARP1 inhibitors attenuated AKT-FOXO3A signaling by activating PHLPP1. → PHLPP1 regulates the sensitivity of cancer cells to PARP1 inhibitors. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

  9. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shuai [State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35205 (United States); Wang, Huibo; Davis, Ben C. [Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35205 (United States); Liang, Jiyong [Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77054 (United States); Cui, Rutao [Department of Dermatology, Boston University School of Medicine, Boston, MA 02118 (United States); Chen, Sai-Juan, E-mail: sjchen@stn.sh.cn [State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Xu, Zhi-Xiang, E-mail: zhi-xiang.xu@ccc.uab.edu [Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35205 (United States)

    2011-08-26

    Highlights: {yields} PARP1 inhibitors cause a cytotoxic effect independent of DNA repair impairment. {yields} PARP1 inhibitors attenuated AKT-FOXO3A signaling by activating PHLPP1. {yields} PHLPP1 regulates the sensitivity of cancer cells to PARP1 inhibitors. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

  10. Methylglyoxal mediates adipocyte proliferation by increasing phosphorylation of Akt1.

    Directory of Open Access Journals (Sweden)

    Xuming Jia

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

  11. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro

    Directory of Open Access Journals (Sweden)

    Ji Young Park

    2012-01-01

    (2.5–20 μg/mL inhibited ADP-induced platelet aggregation. While PCME diminished [Ca2+]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology.

  12. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.

    Science.gov (United States)

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J

    2016-05-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

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

    Science.gov (United States)

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

    2002-01-01

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

  14. AB044. AGE/RAGE/Akt pathway contributes to prostate cancer cell proliferation by promoting Rb phosphorylation and degradation

    Science.gov (United States)

    Bao, Jiming; Bao, Yawei; Zhao, Shanchao; He, Minyi; Luo, Haihua; Ren, Zhonglu; Lv, Yongjie; Hong, Yingqia

    2016-01-01

    Objective Metabolomic research has revealed that metabolites play an important role in prostate cancer development and progression. Previous studies have suggested that prostate cancer cell proliferation is induced by advanced glycation end products (AGEs) exposure, but the mechanism of this induction remains unknown. This study aim to investigate the molecular mechanisms underlying the proliferative response of prostate cancer cell to the interaction of AGEs and the receptor for advanced glycation end products (RAGE). Methods To investigate this mechanism, we used Western blotting to evaluate the responses of the retinoblastoma (Rb), p-Rb and PI3K/Akt pathway to AGEs stimulation. We also examined the effect of knocking down Rb and blocking the PI3K/Akt pathway on AGEs induced PC-3 cell proliferation. Results Our results indicated that AGE-RAGE interaction enhanced Rb phosphorylation and subsequently decreased total Rb levels. Bioinformatics analysis further indicated a negative correlation between RAGE and RB1 expression in prostate cancer tissue. Furthermore, we observed that AGEs stimulation activated the PI3K/Akt signaling pathway and that blocking PI3K/Akt signaling abrogated AGEs-induced cell proliferation. Conclusions We report, for the first time, that AGE-RAGE interaction enhances prostate cancer cell proliferation by phosphorylation of Rb via the PI3K/Akt signaling pathway.

  15. Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition

    OpenAIRE

    Katayama, Kazuhiro; Fujita, Naoya; Tsuruo, Takashi

    2005-01-01

    The serine/threonine kinase Akt is known to promote cell growth by regulating the cell cycle in G1 phase through activation of cyclin/Cdk kinases and inactivation of Cdk inhibitors. However, how the G2/M phase is regulated by Akt remains unclear. Here, we show that Akt counteracts the function of WEE1Hu. Inactivation of Akt by chemotherapeutic drugs or the phosphatidylinositide-3-OH kinase inhibitor LY294002 induced G2/M arrest together with the inhibitory phosphorylation of Cdc2. Because the...

  16. PKCη is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    International Nuclear Information System (INIS)

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKCη isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKCη-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKCη-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKCη exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKCη and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKCη expression, suggesting that PKCη acts through a different route to increase cell survival. Hence, our studies show that PKCη provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  17. PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta, E-mail: etta@bgu.ac.il

    2012-04-15

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  18. KIF14 Promotes AKT Phosphorylation and Contributes to Chemoresistance in Triple-Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    Stina M. Singel

    2014-03-01

    Full Text Available Despite evidence that kinesin family member 14 (KIF14 can serve as a prognostic biomarker in various solid tumors, how it contributes to tumorigenesis remains unclear. We observed that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in estrogen receptor–negative/progesterone receptor–negative/human epidermal growth factor receptor 2-negative, “triple-negative” breast cancers (TNBC. To investigate the oncogenic role of KIF14, we used noncancerous human mammary epithelial cells and ectopically expressed KIF14 and found increased proliferative capacity, increased anchorage-independent grown in vitro, and increased resistance to docetaxel but not to doxorubicin, carboplatin, or gemcitabine. Seventeen benign breast biopsies of BRCA1 or BRCA2 mutation carriers showed increased KIF14 mRNA expression by fluorescence in situ hybridization compared to controls with no known mutations in BRCA1 or BRCA2, suggesting increased KIF14 expression as a biomarker of high-risk breast tissue. Evaluation of 34 cases of locally advanced TNBC showed that KIF14 expression significantly correlates with chemotherapy-resistant breast cancer. KIF14 knockdown also correlates with decreased AKT phosphorylation and activity. Live-cell imaging confirmed an insulin-induced temporal colocalization of KIF14 and AKT at the plasma membrane, suggesting a potential role of KIF14 in promoting activation of AKT. An experimental small-molecule inhibitor of KIF14 was then used to evaluate the potential anticancer benefits of downregulating KIF14 activity. Inhibition of KIF14 shows a chemosensitizing effect and correlates with decreasing activation of AKT. Together, these findings show an early and critical role for KIF14 in the tumorigenic potential of TNBC, and therapeutic targeting of KIF14 is feasible and effective for TNBC.

  19. KIF14 promotes AKT phosphorylation and contributes to chemoresistance in triple-negative breast cancer.

    Science.gov (United States)

    Singel, Stina M; Cornelius, Crystal; Zaganjor, Elma; Batten, Kimberly; Sarode, Venetia R; Buckley, Dennis L; Peng, Yan; John, George B; Li, Hsiao C; Sadeghi, Navid; Wright, Woodring E; Lum, Lawrence; Corson, Timothy W; Shay, Jerry W

    2014-03-01

    Despite evidence that kinesin family member 14 (KIF14) can serve as a prognostic biomarker in various solid tumors, how it contributes to tumorigenesis remains unclear. We observed that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor 2-negative, "triple-negative" breast cancers (TNBC). To investigate the oncogenic role of KIF14, we used noncancerous human mammary epithelial cells and ectopically expressed KIF14 and found increased proliferative capacity, increased anchorage-independent grown in vitro, and increased resistance to docetaxel but not to doxorubicin, carboplatin, or gemcitabine. Seventeen benign breast biopsies of BRCA1 or BRCA2 mutation carriers showed increased KIF14 mRNA expression by fluorescence in situ hybridization compared to controls with no known mutations in BRCA1 or BRCA2, suggesting increased KIF14 expression as a biomarker of high-risk breast tissue. Evaluation of 34 cases of locally advanced TNBC showed that KIF14 expression significantly correlates with chemotherapy-resistant breast cancer. KIF14 knockdown also correlates with decreased AKT phosphorylation and activity. Live-cell imaging confirmed an insulin-induced temporal colocalization of KIF14 and AKT at the plasma membrane, suggesting a potential role of KIF14 in promoting activation of AKT. An experimental small-molecule inhibitor of KIF14 was then used to evaluate the potential anticancer benefits of downregulating KIF14 activity. Inhibition of KIF14 shows a chemosensitizing effect and correlates with decreasing activation of AKT. Together, these findings show an early and critical role for KIF14 in the tumorigenic potential of TNBC, and therapeutic targeting of KIF14 is feasible and effective for TNBC. PMID:24784001

  20. Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177

    Science.gov (United States)

    Walther, Stefanie; Pluteanu, Florentina; Renz, Susanne; Nikonova, Yulia; Maxwell, Joshua T.; Yang, Li-Zhen; Schmidt, Kurt; Edwards, Joshua N.; Wakula, Paulina; Groschner, Klaus; Maier, Lars S.; Spiess, Joachim; Blatter, Lothar A.; Pieske, Burkert

    2014-01-01

    Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca2+-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling. PMID:25015964

  1. Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

    Directory of Open Access Journals (Sweden)

    Hong Wa Yung

    Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

  2. Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    OpenAIRE

    Nacusi Lucas P; Sheaff Robert J

    2006-01-01

    Abstract Background p27kip1 (p27) is a multifunctional protein implicated in regulation of cell cycling, signal transduction, and adhesion. Its activity is controlled in part by Phosphatylinositol-3-Kinase (PI3K)/Akt1 signaling, and disruption of this regulatory connection has been identified in human breast cancers. The serine/threonine protein kinase Akt1 directly phosphorylates p27, so identifying the modified residue(s) is essential for understanding how it regulates p27 function. Various...

  3. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro.

    Science.gov (United States)

    Park, Ji Young; Hong, Mei; Jia, Qi; Lee, Young-Chul; Yayeh, Taddesse; Hyun, Eujin; Kwak, Dong-Mi; Cho, Jae Youl; Rhee, Man Hee

    2012-01-01

    Pistacia chinensis (Chinese pistache) is a widely grown plant in southern China where the galls extract is a common practice in folk medicine. However, extracts from this plant have never been attempted for their cardiovascular protective effects in experimental setting. Here therefore we aimed to investigate the antiplatelet activity of Pistacia chinensis methanolic extract (PCME) in ADP stimulated rat platelets in vitro. PCME (2.5-20 μg/mL) inhibited ADP-induced platelet aggregation. While PCME diminished [Ca(2+)]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology. PMID:22899962

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

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Zhen [Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Gan, Ye-Hua, E-mail: kqyehuagan@bjmu.edu.cn [Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China)

    2015-05-01

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

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

    International Nuclear Information System (INIS)

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

  6. COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer

    Directory of Open Access Journals (Sweden)

    Goodman Julie E

    2010-11-01

    Full Text Available Abstract Background Inducible cyclooxgenase-2 (COX-2 is commonly overexpressed in breast tumors and is a target for cancer therapy. Here, we studied the association of COX-2 with breast cancer survival and how this association is influenced by tumor estrogen and HER2 receptor status and Akt pathway activation. Methods Tumor COX-2, HER2 and estrogen receptor α (ER expression and phosphorylation of Akt, BAD, and caspase-9 were analyzed immunohistochemically in 248 cases of breast cancer. Spearman's correlation and multivariable logistic regression analyses were used to examine the relationship between COX-2 and tumor characteristics. Kaplan-Meier survival and multivariable Cox proportional hazards regression analyses were used to examine the relationship between COX-2 and disease-specific survival. Results COX-2 was significantly associated with breast cancer outcome in ER-negative [Hazard ratio (HR = 2.72; 95% confidence interval (CI, 1.36-5.41; comparing high versus low COX-2] and HER2 overexpressing breast cancer (HR = 2.84; 95% CI, 1.07-7.52. However, the hazard of poor survival associated with increased COX-2 was highest among patients who were both ER-negative and HER2-positive (HR = 5.95; 95% CI, 1.01-34.9. Notably, COX-2 expression in the ER-negative and HER2-positive tumors correlated significantly with increased phosphorylation of Akt and of the two Akt targets, BAD at Ser136 and caspase-9 at Ser196. Conclusions Up-regulation of COX-2 in ER-negative and HER2-positive breast tumors is associated with Akt pathway activation and is a marker of poor outcome. The findings suggest that COX-2-specific inhibitors and inhibitors of the Akt pathway may act synergistically as anticancer drugs in the ER-negative and HER2-positive breast cancer subtype.

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

    Science.gov (United States)

    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. PMID:22345431

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

    Science.gov (United States)

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

    2016-06-16

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

  9. The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation.

    Science.gov (United States)

    Cerniglia, George J; Dey, Souvik; Gallagher-Colombo, Shannon M; Daurio, Natalie A; Tuttle, Stephen; Busch, Theresa M; Lin, Alexander; Sun, Ramon; Esipova, Tatiana V; Vinogradov, Sergei A; Denko, Nicholas; Koumenis, Constantinos; Maity, Amit

    2015-08-01

    Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. PMID:25995437

  10. Sustained oxidative stress causes late acute renal failure via duplex regulation on p38 MAPK and Akt phosphorylation in severely burned rats.

    Directory of Open Access Journals (Sweden)

    Yafei Feng

    Full Text Available BACKGROUND: Clinical evidence indicates that late acute renal failure (ARF predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved. MATERIALS AND METHODS: Rats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area. Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn. RESULTS: Renal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn. CONCLUSIONS: These results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation.

  11. Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells.

    Science.gov (United States)

    Hosokawa, Hiroyuki; Tanaka, Tomoaki; Endo, Yusuke; Kato, Miki; Shinoda, Kenta; Suzuki, Akane; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I; Nakayama, Toshinori

    2016-01-01

    Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells. PMID:27053161

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

    Science.gov (United States)

    Zhen, Jiaojiao; Ai, Zhilong

    2016-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Houbao [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Xu, Wangyang [Department of Clinical Laboratories, Ninth People’s Hospital, SJTUSM, Shanghai 200011 (China); Zhang, Hongxin [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Liu, Jianbing [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Shanghai Research Center for Model Organisms, Shanghai 201203 (China); Xu, Haimin [Department of Pathology, Rui-Jin Hospital, SJTUSM, Shanghai 200025 (China); Lu, Shunyuan; Dang, Suying [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Kuang, Ying [Shanghai Research Center for Model Organisms, Shanghai 201203 (China); Jin, Xiaolong [Department of Pathology, Rui-Jin Hospital, SJTUSM, Shanghai 200025 (China); Wang, Zhugang, E-mail: zhugangw@shsmu.edu.cn [State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Rui-Jin Hospital and Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025 (China); Shanghai Research Center for Model Organisms, Shanghai 201203 (China)

    2013-08-16

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

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

    International Nuclear Information System (INIS)

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

  16. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei, E-mail: fchen@wayne.edu

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  17. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    International Nuclear Information System (INIS)

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H2O2, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H2O2. Furthermore, both arsenic and H2O2 were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As3+) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As3+-induced EZH2 phosphorylation. • As3+ induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm

  18. Does progesterone show neuroprotective effects on traumatic brain injury through increasing phosphorylation of Akt in the hippocampus?

    Institute of Scientific and Technical Information of China (English)

    Richard Justin Garling; Lora Talley Watts; Shane Sprague; Lauren Fletcher; David F Jimenez; Murat Digicaylioglu

    2014-01-01

    There are currently no federally approved neuroprotective agents to treat traumatic brain injury. Progesterone, a hydrophobic steroid hormone, has been shown in recent studies to exhibit neu-roprotective effects in controlled cortical impact rat models. Akt is a protein kinase known to play a role in cell signaling pathways that reduce edema, inlfammation, apoptosis, and promote cell growth in the brain. This study aims to determine if progesterone modulates the phosphor-ylation of Aktvia its threonine 308 phosphorylation site. Phosphorylation at the threonine 308 site is one of several sites responsible for activating Akt and enabling the protein kinase to carry out its neuroprotective effects. To assess the effects of progesterone on Akt phosphorylation, C57BL/6 mice were treated with progesterone (8 mg/kg) at 1 (intraperitonally), 6, 24, and 48 hours (subcutaneously) post closed-skull traumatic brain injury. The hippocampus was harvest-ed at 72 hours post injury and prepared for western blot analysis. Traumatic brain injury caused a signiifcant decrease in Akt phosphorylation compared to sham operation. However, mice treat-ed with progesterone following traumatic brain injury had an increase in phosphorylation of Akt compared to traumatic brain injury vehicle. Our ifndings suggest that progesterone is a viable treatment option for activating neuroprotective pathways after traumatic brain injury.

  19. Effects of Zinc on Glucose Consumption and AKT/GSK3β Phosphorylation in L6 Myotubes

    Institute of Scientific and Technical Information of China (English)

    Hui-zi LU; Yun-tang WU; Zhong SUN; Yong-zhe LIU; Yong-ming WANG; Qian SANG; Xin-yan LIU

    2014-01-01

    ObjectiveTo investigate the effects of zinc on glucose consumption in normal and insulin-resistant L6 myotubes and elucidate its association with AKT/GSK3β phosphorylation, two key components in the insulin-signaling pathway.Methods The insulin-resistant cell model was prepared by treating L6 myotubes with 0.4mmol/L palmitic acid for 24h and then exposed to different concentrations of zinc (0, 10, 20, 50, 100μmol/L) in the presence or absence of insulin (100 nmol/L) for 3h. Glucose consumption was determined by glucose oxidase method. AKT /GSK3β phosphorylation was detected by Western blotting method.ResultsIn normal L6 myotubes, zinc (10-50μmol/L) alone could significantly increase glucose consumption. In the presence or absence of insulin, zinc significantly enhanced AKT/GSK3β phosphorylation. In insulin-resistant L6 myotubes, zinc (10-50μmol/L) could increase glucose consumption and GSK3β phosphorylation, which was accompanied by enhanced AKT phosphorylation in the presence of insulin.ConclusionCollectively, these results showed that zinc at the concentrations of 10-50μmol/L could increase glucose consumption in L6 myotubes. The mechanism was related to the activation of the insulin signaling pathway by zinc through AKT/GSK3β phosphorylation.

  20. Interference with Akt signaling pathway contributes curcumin-induced adipocyte insulin resistance.

    Science.gov (United States)

    Zhang, Deling; Zhang, Yemin; Ye, Mao; Ding, Youming; Tang, Zhao; Li, Mingxin; Zhou, Yu; Wang, Changhua

    2016-07-01

    Previous study has shown that curcumin directly or indirectly suppresses insulin signaling in 3T3-L1 adipocytes. However, the underlying mechanism remains unclear. Here we experimentally demonstrate that curcumin inhibited the ubiquitin-proteasome system (UPS) function, activated autophagy, and reduced protein levels of protein kinase B (Akt) in a dose- and time-dependent manner in 3T3-L1 adipocytes, accompanied with attenuation of insulin-stimulated Akt phosphorylation, plasma membrane translocation of glucose transporter type 4 (GLUT4), and glucose uptake. These in vitro inhibitory effects of curcumin on Akt protein expression and insulin action were reversed by pharmacological and genetic inhibition of autophagy but not by inhibition of the UPS and caspases. In addition, Akt reduction in adipose tissues of mice treated with curcumin could be recovered by administration of autophagy inhibitor bafilomycin A1 (BFA). This new finding provides a novel mechanism by which curcumin induces insulin resistance in adipocytes. PMID:27113027

  1. Luteolin Inhibits Angiotensin II-Stimulated VSMC Proliferation and Migration through Downregulation of Akt Phosphorylation

    Directory of Open Access Journals (Sweden)

    Tongda Xu

    2015-01-01

    Full Text Available Luteolin is a naturally occurring flavonoid found in many plants that possesses cardioprotective properties. The purpose of this study was to elucidate the effect of luteolin on vascular smooth muscle cells (VSMCs proliferation and migration induced by Angiotensin II (Ang II and to investigate the mechanism(s of action of this compound. Rat VSMCs were cultured in vitro, and the proliferation and migration of these cells following Ang II stimulation were monitored. Different doses of luteolin were added to VSMC cultures, and the proliferation and migration rate were observed by MTT and Transwell chamber assays, respectively. In addition, the expressions of p-Akt (308, p-Akt (473, and proliferative cell nuclear antigen (PCNA in VSMCs were monitored by Western blotting. This study demonstrated that luteolin has an inhibitory effect on Ang II-induced VSMC proliferation and migration. Further, the levels of p-Akt (308, p-Akt (473, and PCNA were reduced in VSMCs treated with both Ang II and luteolin compared to VSMCs treated with only Ang II. These findings strongly suggest that luteolin inhibits Ang II-stimulated proliferation and migration of VSMCs, which is partially due to downregulation of the Akt signaling pathway.

  2. Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2

    International Nuclear Information System (INIS)

    Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers. Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients. Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR. Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours

  3. Amoxicillin and amoxicillin/clavulanate reduce ethanol intake and increase GLT-1 expression as well as AKT phosphorylation in mesocorticolimbic regions.

    Science.gov (United States)

    Goodwani, Sunil; Rao, P S S; Bell, Richard L; Sari, Youssef

    2015-10-01

    Studies have shown that administration of the β-lactam antibiotic ceftriaxone (CEF) attenuates ethanol consumption and cocaine seeking behavior as well as prevents ethanol-induced downregulation of glutamate transporter 1 (GLT-1) expression in central reward brain regions. However, it is not known if these effects are compound-specific. Therefore, the present study examined the effects of two other β-lactam antibiotics, amoxicillin (AMOX) and amoxicillin/clavulanate (Augmentin, AUG), on ethanol drinking, as well as GLT-1 and phosphorylated-AKT (pAKT) levels in the nucleus accumbens (Acb) and medial prefrontal cortex (mPFC) of alcohol-preferring (P) rats. P rats were exposed to free-choice of ethanol (15% and 30%) for five weeks and were given five consecutive daily i.p. injections of saline vehicle, 100 mg/kg AMOX or 100mg/kg AUG. Both compounds significantly decreased ethanol intake and significantly increased GLT-1 expression in the Acb. AUG also increased GLT-1 expression in the mPFC. Results for changes in pAKT levels matched those for GLT-1, indicating that β-lactam antibiotic-induced reductions in ethanol intake are negatively associated with increases in GLT-1 and pAKT levels within two critical brains regions mediating drug reward and reinforcement. These findings add to a growing literature that pharmacological increases in GLT-1 expression are associated with decreases in ethanol intake and suggest that one mechanism mediating this effect may be increased phosphorylation of AKT. Thus, GLT-1 and pAKT may serve as molecular targets for the treatment of alcohol and drug abuse/dependence. PMID:26168897

  4. Akt-mediated regulation of antidepressant-sensitive serotonin transporter function, cell-surface expression and phosphorylation

    DEFF Research Database (Denmark)

    Rajamanickam, Jeyaganesh; Annamalai, Balasubramaniam; Rahbek-Clemmensen, Troels; Sundaramurthy, Santhanalakshmi; Gether, Ulrik; Jayanthi, Lankupalle D; Ramamoorthy, Sammanda

    2015-01-01

    The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary targets for antidepressants for therapeutic intervention of mood disorders. Our previous...... studies have identified the involvement of several signalling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether Akt/PKB (protein kinase) regulates SERT function is not known. In the present study, we made the novel observation that inhibition of...... basal phosphorylation. Our results provide evidence that Akt regulates SERT function and cell-surface expression by regulating the intracellular SERT distribution and plasma membrane availability, which perhaps may be linked to SERT phosphorylation state. Thus any changes in the activation of Akt and...

  5. Salt stress-induced protein phosphorylation

    International Nuclear Information System (INIS)

    Protein phosphorylation induced by salt stress in tomato germinating seeds were investigated by two-dimensional polyacrilamide gel electrophoresis of proteins labeled in vivo with (32P)-Phosphate. NaCl induced the phosphorylation of a 14 Kd polypeptide. Pulse-chase experiments revealed that the phosphorylated molecules of this polypeptide are only stable while the stress is present. Phosphorylated 14 Kd polypeptides could be detected in radicles of salt-shocked seedlings after 6 hours stress period. 14 Kd polypeptide phosphorylation was also observed in seeds germinating in the presence of abscisic acid (ABA). The amount of phosphorylated 14 Kd polypeptide was significantly increased in seeds treated simultaneously with NaCl and ABA

  6. Tamoxifen-induced cytotoxicity in breast cancer cells is mediated by glucose-regulated protein 78 (GRP78) via AKT (Thr308) regulation.

    Science.gov (United States)

    Pujari, Radha; Jose, Jemy; Bhavnani, Varsha; Kumar, Natesh; Shastry, Padma; Pal, Jayanta K

    2016-08-01

    Glucose regulated protein 78 (GRP78) has recently been suggested to be associated with drug resistance in breast cancer patients. However, the precise role of GRP78 in drug resistance and the involved signaling pathways are not clearly understood. In the present study, we show that among a panel of drugs, namely Paclitaxel (TAX), Doxorubicin (DOX), 5-fluorouracil (5-FU), UCN-01 and Tamoxifen (TAM) used, TAM alone up-regulated the expression of GRP78 significantly and induced apoptosis in MCF-7 and MDA-MB-231 cells. Interestingly, inhibition of GRP78 by a specific pharmacological inhibitor, VER-155008 augmented TAM-induced apoptosis, and overexpression of GRP78 rendered the cells resistant to TAM-induced cell death suggesting a role for GRP78 in TAM-induced cytotoxicity. Mechanistically, the expression of phosphorylated AKT as determined by Western blot analyses revealed that TAM selectively upregulated phosphorylation of AKT at Thr308 but not at Ser473, and siRNA silencing of GRP78 resulted in inhibition of AKT phosphorylation at Thr308 but not at Ser473. Further, a GRP78 inhibitor, VER155008 inhibited TAM-induced phosphorylation of GSK3β, a downstream substrate of AKT. These results, thus suggests a role for GRP78 in TAM-induced AKT activation. Additionally, co-localization studies by immunofluorescence, and immunoprecipitation experiments demonstrated a complex formation of AKT and GRP78. Furthermore, in glucose-free medium, the cells were sensitized to TAM-induced cell death that was associated with reduced AKT phosphorylation at Thr308, thus strengthening the association of AKT regulation with drug response. Collectively, our findings identify a role of GRP78 in AKT regulation in response to TAM in breast cancer cells. PMID:27262235

  7. Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells

    Directory of Open Access Journals (Sweden)

    Zhang Chen-Ou

    2010-12-01

    Full Text Available Abstract Background Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4. Because synthetic asialo-APF (as-APF has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells. Methods T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β, β-catenin, p53, and matrix metalloproteinase 2 (MMP2 mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR. Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot. Results T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or

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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jintao; Zhu, Dexiao; Zhang, Jing; Li, Guibao [Department of Anatomy, School of Medicine, Shandong University, Jinan, Shandong, 250012 (China); Liu, Zengxun [Department of Psychiatry, School of Medicine, Shandong University, Jinan, Shandong, 250012 China (China); Sun, Jinhao, E-mail: sunjinhao@gmail.com [Department of Anatomy, School of Medicine, Shandong University, Jinan, Shandong, 250012 (China)

    2015-09-25

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

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

    International Nuclear Information System (INIS)

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

  10. Leucine minimizes denervation-induced skeletal muscle atrophy of rats through akt/mtor signaling pathways

    Directory of Open Access Journals (Sweden)

    Carolina Barbosa Ribeiro

    2015-03-01

    Full Text Available The aim of the present study was to evaluate the effect of leucine treatment (0.30 mM on muscle weight and signaling of myoproteins related to synthesis and degradation pathways of soleus muscle following seven days of complete sciatic nerve lesion.Wistar rats (n=24 of 3 to 4 months of age (192 ± 23 g were used. The animals were randomly distributed into four experimental groups (n=6/group: control, treated with leucine (L, denervated (D and denervated treated with leucine (DL.Dependent measures were proteins levels of AKT, AMPK, mTOR, and ACC performed by Western blot. Leucine induced a reduction in the phosphorylation of AMPK (p<0.05 by 16% in the L and by 68% in the DL groups as compared with control group. Denervation increased AMPK by 24% in the D group as compared with the control group (p<0.05. AKT was also modulated by denervation and leucine treatment, highlighted by the elevation of AKT phosphorylation in the D (65%, L (98% and DL (146% groups as compared with the control group (p<0.05. AKT phosphorylation was 49% higher in the D group as compared with the DL group.Furthermore, denervation decreased mTOR phosphorylation by 29% in the D group as compared with the control group. However, leucine treatment induced an increase of 49% in the phosphorylation of mTOR in the L group as compared with the control group, and an increase of 154% in the DL as compared with the D group ( p<0.05. ACC phosphorylation was 20% greater in the D group than the control group. Furthermore, ACC in the soleus was 22% lower in the in the L group and 50% lower in the DL group than the respective control group (p<0.05.In conclusion, leucine treatment minimized the deleterious effects of denervation on rat soleus muscle by increasing anabolic (AKT and mTOR and decreasing catabolic (AMPK pathways. These results may be interesting for muscle recovery following acute denervation, which may contribute to musculoskeletal rehabilitation after denervation.

  11. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    Science.gov (United States)

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Inhibition of PKB/Akt activity involved in apigenin-induced apoptosis in human gastric carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    YUAN LinHong; XIA Wei; ZHAO XiuJuan; ZHANG XiaoHua; ZHANG Ling; WU Kun

    2007-01-01

    Apigenin is a flavonoid widely distributed in fruits and vegetables.It possesses growth inhibitory properties against numerous cancer cell lines.However, the molecular mechanism(s) by which apigenin elicits its effects have not been fully elucidated.Here we studied whether apigenin inhibits growth and induces apoptosis in human gastric carcinoma cells.We showed that the flavonoid inhibited growth of the cells and caused apoptosis, as evidenced by DNA Ladder, cleavage of pro-caspase-3 in a time-dependent manner.Induction of apoptosis was dependent on inhibition of the PKB/Akt activity.We found that while apigenin had no effect on the expression of Akt and Bad, it inhibited specific phosphorylation of the two proteins that are associated with pro-survival mechanisms.We propose that this important flavonoid induces apoptosis in gastric cancer cells by inhibiting Akt activity.Since Akt is often activated in cancers, our findings may have clinical implications.

  14. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer.

    Science.gov (United States)

    Nacerddine, Karim; Beaudry, Jean-Bernard; Ginjala, Vasudeva; Westerman, Bart; Mattiroli, Francesca; Song, Ji-Ying; van der Poel, Henk; Ponz, Olga Balagué; Pritchard, Colin; Cornelissen-Steijger, Paulien; Zevenhoven, John; Tanger, Ellen; Sixma, Titia K; Ganesan, Shridar; van Lohuizen, Maarten

    2012-05-01

    Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis. PMID:22505453

  15. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

    Science.gov (United States)

    Nacerddine, Karim; Beaudry, Jean-Bernard; Ginjala, Vasudeva; Westerman, Bart; Mattiroli, Francesca; Song, Ji-Ying; van der Poel, Henk; Ponz, Olga Balagué; Pritchard, Colin; Cornelissen-Steijger, Paulien; Zevenhoven, John; Tanger, Ellen; Sixma, Titia K.; Ganesan, Shridar; van Lohuizen, Maarten

    2012-01-01

    Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis. PMID:22505453

  16. Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway

    Science.gov (United States)

    Xu, Bing-Can; Long, Hui-Bao; Luo, Ke-Qin

    2016-01-01

    Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice. PMID:27435826

  17. Absence of the GPR37/PAEL receptor impairs striatal Akt and ERK2 phosphorylation, DeltaFosB expression, and conditioned place preference to amphetamine and cocaine.

    Science.gov (United States)

    Marazziti, Daniela; Di Pietro, Chiara; Mandillo, Silvia; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

    2011-06-01

    The orphan G-protein-coupled receptor 37 (GPR37) colocalizes with the dopamine (DA) transporter (DAT) in mouse nigrostriatal presynaptic membranes, and its genetic ablation in homozygous null-mutant (GPR37-KO) mice provokes the marked increase of plasma membrane expression of DAT, alteration of psychostimulant-induced locomotor activity, and reduction of catalepsy induced by DA-receptor antagonists. We report that extracts from GPR37-KO mice displayed biochemical alterations of the nigrostriatal signaling pathways mediated by D1 and D2 dopaminergic receptors. Null-mutant mice showed an increase of the basal phosphorylation level of the D2-regulated Akt kinase. The basal phosphorylation of the D1-activated ERK2 kinase was not altered, but acute treatments with amphetamine or cocaine failed to produce its specific increase, as detected in samples from wild-type littermates. Furthermore, the chronic administration of cocaine to GPR37-KO mice did not increase the expression of the ΔFosB transcription factor isoforms. Consistently, behavioral analysis showed that null-mutant animals did not respond to the incentive properties of amphetamine or cocaine, in conditioned place preference tests. Thus, the lack of GPR37 affects both ERK2- and Akt-mediated striatal signaling pathways, impairing the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs. PMID:21372109

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. We previously demonstrated that phosphorylation of Threonine-308 on Akt (pAkt-...

  19. Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells

    Science.gov (United States)

    Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

    2016-01-01

    Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways. PMID:27143997

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

    Energy Technology Data Exchange (ETDEWEB)

    Roffe, Suzy [Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel); Hagai, Yosey [Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel); Institute of Animal Sciences, Volcani Center, Bet Dagan 50250 (Israel); Pines, Mark [Institute of Animal Sciences, Volcani Center, Bet Dagan 50250 (Israel); Halevy, Orna, E-mail: halevyo@agri.huji.ac.il [Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel)

    2010-04-01

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

  1. SP600125 Induces Src and Type I IGF Receptor Phosphorylation Independent of JNK

    Directory of Open Access Journals (Sweden)

    Qingbin Kong

    2014-09-01

    Full Text Available c-Jun N-terminal kinases (JNK are members of the mitogen-activated protein kinase (MAPK family that have important roles in signal transduction. The small molecule SP600125 is widely used in biochemical studies as a JNK inhibitor. However, recent studies indicate that SP600125 may also act independent of JNK. Here, we report that SP600125 can induce Src, type I insulin-like growth factor receptor (IGF-IR, Akt and Erk1/2 phosphorylation. Notably, these effects are independent of its inhibition of JNK. Inhibition of Src abrogates the stimulation of IGF-IR, Akt and Erk1/2 phosphorylation. IGF-IR knockdown blunts the induction of both Akt and Erk1/2 phosphorylation by SP600125. Moreover, combination of SP600125 and the Src inhibitor saracatinib synergistically inhibits cell proliferation. We conclude that SP600125 can activate Src-IGF-IR-Akt/Erk1/2 signaling pathways independent of JNK.

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

    Science.gov (United States)

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

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway. PMID:27233246

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

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Tadashi [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Nakamura, Shigeo [Department of Chemistry, Nippon Medical School, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-0023 (Japan); Ono, Toshiya; Ui, Sadaharu [Department of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu 400-8511 (Japan); Yagi, Syota; Kagawa, Hiroki [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Watanabe, Hisami [Center of Molecular Biosciences, Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213 (Japan); Ohe, Tomoyuki; Mashino, Tadahiko [Department of Pharmaceutical Sciences, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

    2014-08-15

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

  4. Loss of Akt1 or Akt2 delays mammary tumor onset and suppresses tumor growth rate in MTB-IGFIR transgenic mice

    International Nuclear Information System (INIS)

    Akt is a serine/threonine kinase that mediates signaling downstream of tyrosine kinase receptors like the type I insulin-like growth factor receptor (IGF-IR). In fact, we have previously shown that mammary tumors induced by elevated expression of the IGF-IR are associated with hyperactivation of Akt. However, there are three mammalian isoforms of Akt (Akt1, Akt2 and Akt3) and these isoforms regulate distinct physiologic properties within cells. In this manuscript, the impact of disrupting Akt1 or Akt2 in mammary tumors induced by IGF-IR overexpression were examined to determine whether specific Akt isoforms regulate different aspects of mammary tumorigenesis. Akt1 and Akt2 levels were stably ablated in mammary tumors of MTB-IGFIR transgenic mice by crossing MTB-IGFIR transgenic mice with either Akt1−/− or Akt2−/− mice. Tumor onset, growth rate, and metastasis were determined. Ablation of Akt1 or Akt2 significantly delayed tumor onset and tumor growth rate but did not significantly alter lung metastasis. Despite the absence of Akt1 or Akt2, mammary tumors that developed in the MTB-IGFIR mice maintained detectable levels of phosphorylated Akt. Disruption of Akt1 or Akt2 did not affect cell morphology or the expression of luminal or basal cytokeratins in mammary tumors. Although loss of Akt1 or Akt2 significantly inhibited mammary tumor onset and growth rates the effects were less dramatic than anticipated. Despite the complete loss of Akt1 or Akt2, the level of total phosphorylated Akt remained largely unaffected in the mammary tumors suggesting that loss of one Akt isoform is compensated by enhanced activation of the remaining Akt isoforms. These findings indicate that therapeutic strategies targeting the activation of individual Akt isoforms will prove less effective than simultaneously inhibiting the activity of all three Akt isoforms for the treatment of breast cancer

  5. Activation of Akt1 accelerates carcinogen-induced tumorigenesis in mammary gland of virgin and post-lactating transgenic mice

    International Nuclear Information System (INIS)

    Data from in vivo and in vitro studies suggest that activation of Akt regulates cell survival signaling and plays a key role in tumorigenesis. Hence, transgenic mice were created to explore the oncogenic role of Akt1 in the development of mammary tumors. The transgenic mice were generated by expressing myristoylated-Akt1 (myr-Akt1) under the control of the MMTV-LTR promoter. The carcinogen 7, 12 dimethyl-1,2-benzanthracene (DMBA) was used to induce tumor formation. The MMTV driven myr-Akt1 transgene expression was detected primarily in the mammary glands, uterus, and ovaries. The expression level increased significantly in lactating mice, suggesting that the response was hormone dependent. The total Akt expression level in the mammary gland was also higher in the lactating mice. Interestingly, the expression of MMTVmyr-Akt1 in the ovaries of the transgenic mice caused significant increase in circulating estrogen levels, even at the post-lactation stage. Expression of myr-Akt1 in mammary glands alone did not increase the frequency of tumor formation. However, there was an increased susceptibility of forming mammary tumors induced by DMBA in the transgenic mice, especially in mice post-lactation. Within 34 weeks, DMBA induced mammary tumors in 42.9% of transgenic mice post-lactation, but not in wild-type mice post-lactation. The myr-Akt1 mammary tumors induced by DMBA had increased phosphorylated-Akt1 and showed strong expression of estrogen receptor (ERα) and epidermal growth factor receptor (EGFR). In addition, Cyclin D1 was more frequently up-regulated in mammary tumors from transgenic mice compared to tumors from wild-type mice. Overexpression of Cyclin D1, however, was not completely dependent on activated Akt1. Interestingly, mammary tumors that had metastasized to secondary sites had increased expression of Twist and Slug, but low expression of Cyclin D1. In summary, the MMTVmyr-Akt1 transgenic mouse model could be useful to study mechanisms of ER

  6. p38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis

    International Nuclear Information System (INIS)

    Doxorubicin is the anthracycline with the widest spectrum of antitumor activity, and it has been shown that the antitumor activity is mediated in vivo by selective triggering of apoptosis in proliferating endothelial cells. We studied cultured human endothelial cells and observed that doxorubicin-induced apoptosis was mediated by p38 mitogen-activated protein kinase (MAPK). Doxorubicin-provoked apoptosis was significantly inhibited by expression of dominant negative p38 MAPK or pharmacological inhibition with SB203580. Furthermore, blocking phosphatidylinositol-3-kinase/Akt signaling significantly increased doxorubicin-induced caspase-3 activity and cell death, indicating that Akt is a survival factor in this system. Notably, we also found that doxorubicin-provoked apoptosis included p38 MAPK-mediated inhibition of Akt and Bad phosphorylation. Furthermore, doxorubicin-stimulated phosphorylation of Bad in cells expressing dominant negative p38 MAPK was impeded by the inhibition of PI3-K. In addition to the impact on Bad phosphorylation, doxorubicin-treatment caused p38 MAPK-dependent downregulation of Bcl-xL protein

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Beom Su [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of); Park, Ji-Yun [Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of); Kang, Hyo-Jin [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Hyung-Jin [Department of Microbiology, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Jun, E-mail: omslee@wku.ac.kr [Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Bonecell Biotech Inc., 77, Dunsan-dong, Seo-gu, Daejeon 302-830 (Korea, Republic of)

    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

  9. Increased Phosphorylation of PI3K/Akt/mTOR in the Obstructed Kidney of Rats with Unilateral Ureteral Obstruction

    OpenAIRE

    Ma, Seong Kwon; Joo, Soo Yeon; Kim, Chang Seong; Choi, Joon Seok; Bae, Eun Hui; Lee, JongUn; Kim, Soo Wan

    2013-01-01

    The present study aimed to investigate changes in the mammalian target of rapamycin (mTOR) signaling pathway in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Male Sprague-Dawley rats were unilaterally obstructed by ligation of the left proximal ureter for 7 days. Control rats were treated in the same way except that no ligature was made. The expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, and mTOR were determined in the kidney by semi...

  10. Knockdown of Akt Sensitizes Osteosarcoma Cells to Apoptosis Induced by Cisplatin Treatment

    OpenAIRE

    Changwei Yang; Yushu Bai; Guoyou Zhang; Xiaodong Zhu; Ming Li

    2011-01-01

    Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased ...

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

    International Nuclear Information System (INIS)

    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. 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. 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. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yong; Fang, Shi-ji [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China); Zhu, Li-juan [Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021 (China); Zhu, Lun-qing, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children’s Bone Diseases, The Children’s Hospital Affiliated to Soochow University, Suzhou, Jiangsu 215000 (China); Zhou, Xiao-zhong, E-mail: zhouxz@suda.edu.cn [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China)

    2014-10-24

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

  13. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection

    OpenAIRE

    Hurt, K. Joseph; Musicki, Biljana; Palese, Michael A.; Crone, Julie K.; Becker, Robyn E.; Moriarity, John L.; Snyder, Solomon H.; Burnett, Arthur L.

    2002-01-01

    In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phos...

  14. Akt activation induces hypertrophy without contractile phenotypic maturation in airway smooth muscle

    OpenAIRE

    Ma, Lan; Brown, Melanie; Kogut, Paul; Serban, Karina; Li, Xiaojing; McConville, John; Chen, Bohao; Bentley, J. Kelley; Hershenson, Marc B.; Dulin, Nickolai; Solway, Julian; Camoretti-Mercado, Blanca

    2011-01-01

    Airway smooth muscle (ASM) hypertrophy is a cardinal feature of severe asthma, but the underlying molecular mechanisms remain uncertain. Forced protein kinase B/Akt 1 activation is known to induce myocyte hypertrophy in other muscle types, and, since a number of mediators present in asthmatic airways can activate Akt signaling, we hypothesized that Akt activation could contribute to ASM hypertrophy in asthma. To test this hypothesis, we evaluated whether Akt activation occurs naturally within...

  15. Carvedilol protects bone marrow stem cells against hydrogen peroxide-induced cell death via PI3K-AKT pathway.

    Science.gov (United States)

    Chen, Meihui; Chen, Shudong; Lin, Dingkun

    2016-03-01

    Carvedilol, a nonselective β-adrenergic receptor blocker, has been reported to exert potent anti-oxidative activities. In the present study, we aimed to investigate the effects of carvedilol against hydrogen peroxide (H2O2)-induced bone marrow-derived mesenchymal stem cells (BMSCs) death, which imitate the microenvironment surrounding transplanted cells in the injured spinal cord in vitro. Carvedilol significantly reduced H2O2-induced reactive oxygen species production, apoptosis and subsequent cell death. LY294002, the PI3K inhibitor, blocked the protective effects and up-regulation of Akt phosphorylation of carvedilol. Together, our results showed that carvedilol protects H2O2-induced BMSCs cell death partly through PI3K-Akt pathway, suggesting carvedilol could be used in combination with BMSCs for the treatment of spinal cord injury by improving the cell survival and oxidative stress microenvironments. PMID:26898450

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

    Science.gov (United States)

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

    2016-06-01

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

  17. Right-To-Left Ventricular Differences in the Expression of Mitochondrial Hexokinase and Phosphorylation of Akt

    Czech Academy of Sciences Publication Activity Database

    Wasková-Arnoštová, P.; Elsnicová, B.; Kašparová, D.; Šebesta, O.; Novotný, J.; Neckář, Jan; Kolář, František; Žurmanová, J.

    2013-01-01

    Roč. 31, č. 1 (2013), s. 66-79. ISSN 1015-8987 R&D Projects: GA AV ČR(CZ) IAAX01110901 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : hexokinase isoforms * akt kinase * left ventricle * right ventricle * mitochondria co-localization Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 3.550, year: 2013

  18. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    OpenAIRE

    Wang, Shuai; Wang, Huibo; Davis, Ben C.; Liang, Jiyong; Cui, Rutao; Chen, Sai-Juan; Xu, Zhi-Xiang

    2011-01-01

    Poly (ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphor...

  19. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling

    Science.gov (United States)

    Campos, Ludmila S.; Rodriguez, Yamila I.; Leopoldino, Andreia M.; Hait, Nitai C.; Lopez Bergami, Pablo; Castro, Melina G.; Sanchez, Emilse S.; Maceyka, Michael

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  20. Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: Involvement of Src/EGFR/PI3-K/Akt pathway

    International Nuclear Information System (INIS)

    In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-κB in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS

  1. Helicobacter pylori CagA induces tumor suppressor gene hypermethylation by upregulating DNMT1 via AKT-NFκB pathway in gastric cancer development

    Science.gov (United States)

    Wang, He-xiao; Zhao, Wei; Li, Jian-fang; Su, Li-ping; Shao, Zhifeng; Zhao, Xiaodong; Zhu, Zheng-gang; Yan, Min; Liu, Bingya

    2016-01-01

    Methylation of CpG islands in tumor suppressor gene prompter is one of the most characteristic abnormalities in Helicobacter pylori (HP)-associated gastric carcinoma (GC). Here, we investigated the pathogenic and molecular mechanisms underlying hypermethylation of tumor suppressor genes in HP induced GC development. We found that tumor suppressor genes hypermethylation, represented by MGMT, positively correlated with CagA in clinical specimens, gastric tissues from HP infected C57 mice and GC cell lines transfected by CagA or treated by HP infection. CagA enhanced PDK1 and AKT interaction and increased AKT phosphorylation. The P-AKT subsequent activated NFκB, which then bound to DNMT1 promoter and increased its expression. Finally, the upregulated DNMT1 promoted tumor suppressor genes hypermethylation with MGMT as a representative. In conclusion, CagA increased tumor suppressor genes hypermethylation via stimulating DNMT1 expression through the AKT-NFκB pathway. PMID:26848521

  2. Helicobacter pylori CagA induces tumor suppressor gene hypermethylation by upregulating DNMT1 via AKT-NFκB pathway in gastric cancer development.

    Science.gov (United States)

    Zhang, Bao-Gui; Hu, Lei; Zang, Ming De; Wang, He-Xiao; Zhao, Wei; Li, Jian-Fang; Su, Li-Ping; Shao, Zhifeng; Zhao, Xiaodong; Zhu, Zheng-Gang; Yan, Min; Liu, Bingya

    2016-03-01

    Methylation of CpG islands in tumor suppressor gene prompter is one of the most characteristic abnormalities in Helicobacter pylori (HP)-associated gastric carcinoma (GC). Here, we investigated the pathogenic and molecular mechanisms underlying hypermethylation of tumor suppressor genes in HP induced GC development. We found that tumor suppressor genes hypermethylation, represented by MGMT, positively correlated with CagA in clinical specimens, gastric tissues from HP infected C57 mice and GC cell lines transfected by CagA or treated by HP infection. CagA enhanced PDK1 and AKT interaction and increased AKT phosphorylation. The P-AKT subsequent activated NFκB, which then bound to DNMT1 promoter and increased its expression. Finally, the upregulated DNMT1 promoted tumor suppressor genes hypermethylation with MGMT as a representative. In conclusion, CagA increased tumor suppressor genes hypermethylation via stimulating DNMT1 expression through the AKT-NFκB pathway. PMID:26848521

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

    Science.gov (United States)

    Li, Fang; Malik, Kafait U

    2005-03-01

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

  4. Protein Kinase B (Akt) Promotes Pathological Angiogenesis in Murine Model of Oxygen-Induced Retinopathy

    International Nuclear Information System (INIS)

    Akt, or protein kinase B, is an important signaling molecule that modulates many cellular processes such as cell growth, survival, and metabolism. However, the vivo roles and effectors of Akt in retinal angiogenesis are not explicitly clear. We therefore detected the expression of Akt using Western blotting or RT-PCR technologies in an animal model of oxygen-induced retinopathy, and investigated the effects of recombinant Akt on inhibiting vessels loss and Akt inhibitor on suppressing experimental retinal neovascularization in this model. We showed that in the hyperoxic phase of oxygen-induced retinopathy, the expression of Akt was greatly suppressed. In the hypoxic phase, the expression of Akt was increased dramatically. No significant differences were found in normoxic groups. Compared with control groups, administration of the recombinant Akt in the first phase of retinopathy markedly reduced capillary-free areas, while the administration of the Akt inhibitor in the second phase of retinopathy significantly decreased retinal neovascularization but capillary-free areas. These results indicate that Akt play a critical role in the pathological process (vessels loss and neovascularization) of mouse model of oxygen-induced retinopathy, which may provide a valubale therapeutic tool for ischemic-induced retinal diseases

  5. Dietary Lycium barbarum Polysaccharide Induces Nrf2/ARE Pathway and Ameliorates Insulin Resistance Induced by High-Fat via Activation of PI3K/AKT Signaling

    Directory of Open Access Journals (Sweden)

    Yi Yang

    2014-01-01

    Full Text Available Lycium barbarum polysaccharide (LBP, an antioxidant from wolfberry, displays the antioxidative and anti-inflammatory effects on experimental models of insulin resistance in vivo. However, the effective mechanism of LBP on high-fat diet-induced insulin resistance is still unknown. The objective of the study was to investigate the mechanism involved in LBP-mediated phosphatidylinositol 3-kinase (PI3K/AKT/Nrf2 axis against high-fat-induced insulin resistance. HepG2 cells were incubated with LBP for 12 hrs in the presence of palmitate. C57BL/6J mice were fed a high-fat diet supplemented with LBP for 24 weeks. We analyzed the expression of nuclear factor-E2-related factor 2 (Nrf2, Jun N-terminal kinases (JNK, and glycogen synthase kinase 3β (GSK3β involved in insulin signaling pathway in vivo and in vitro. First, LBP significantly induced phosphorylation of Nrf2 through PI3K/AKT signaling. Second, LBP obviously increased detoxification and antioxidant enzymes expression and reduced reactive oxygen species (ROS levels via PI3K/AKT/Nrf2 axis. Third, LBP also regulated phosphorylation levels of GSK3β and JNK through PI3K/AKT signaling. Finally, LBP significantly reversed glycolytic and gluconeogenic genes expression via the activation of Nrf2-mediated cytoprotective effects. In summary, LBP is novel antioxidant against insulin resistance induced by high-fat diet via activation of PI3K/AKT/Nrf2 pathway.

  6. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  7. Withaferin-A suppress AKT induced tumor growth in colorectal cancer cells.

    Science.gov (United States)

    Suman, Suman; Das, Trinath P; Sirimulla, Suman; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2016-03-22

    The oncogenic activation of AKT gene has emerged as a key determinant of the aggressiveness of colorectal cancer (CRC); hence, research has focused on targeting AKT signaling for the treatment of advanced stages of CRC. In this study, we explored the anti-tumorigenic effects of withaferin A (WA) on CRC cells overexpressing AKT in preclinical (in vitro and in vivo) models. Our results indicated that WA, a natural compound, resulted in significant inhibition of AKT activity and led to the inhibition of cell proliferation, migration and invasion by downregulating the epithelial to mesenchymal transition (EMT) markers in CRC cells overexpressing AKT. The oral administration of WA significantly suppressed AKT-induced aggressive tumor growth in a xenograft model. Molecular analysis revealed that the decreased expression of AKT and its downstream pro-survival signaling molecules may be responsible for tumor inhibition. Further, significant inhibition of some important EMT markers, i.e., Snail, Slug, β-catenin and vimentin, was observed in WA-treated human CRC cells overexpressing AKT. Significant inhibition of micro-vessel formation and the length of vessels were evident in WA-treated tumors, which correlated with a low expression of the angiogenic marker RETIC. In conclusion, the present study emphasizes the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in CRC cells and suggests that WA may overcome AKT-induced cell proliferation and tumor growth in CRC. PMID:26883103

  8. Investigating the function of Akt by tet-off inducible ex- pression system

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A tet-off inducible cell line named BBT derived from BA/F3b cell line was constructed and the effect of this inducible expression system was significant when detected by tet-off responded luciferase reporter gene assay. Then tet-off responded Akt expression plasmid was transfected into BBT cells, and the stable cell lines were screened. The result of Northern blot showed that the expression of akt was signifi-cantly inducible. The clone with the best inducible effect was selected and named BBA for investigating the function of Akt. We found that Akt could significantly inhibit zinc-induced decrease of cell viability when assayed by MTT method. And the flow cytometric analysis showed that Akt could markedly repress zinc-induced apoptosis.

  9. Angiotensin II down-regulates nephrin–Akt signaling and induces podocyte injury: roleof c-Abl

    Science.gov (United States)

    Yang, Qian; Ma, Yiqiong; Liu, Yipeng; Liang, Wei; Chen, Xinghua; Ren, Zhilong; Wang, Huiming; Singhal, Pravin C.; Ding, Guohua

    2016-01-01

    Recent studies have shown that nephrin plays a vital role in angiotensin II (Ang II)–induced podocyte injury and thus contributes to the onset of proteinuria and the progression of renal diseases, but its specific mechanism remains unclear. c-Abl is an SH2/SH3 domain–containing nonreceptor tyrosine kinase that is involved in cell survival and regulation of the cytoskeleton. Phosphorylated nephrin is able to interact with molecules containing SH2/SH3 domains, suggesting that c-Abl may be a downstream molecule of nephrin signaling. Here we report that Ang II–infused rats developed proteinuria and podocyte damage accompanied by nephrin dephosphorylation and minimal interaction between nephrin and c-Abl. In vitro, Ang II induced podocyte injury and nephrin and Akt dephosphorylation, which occurred in tandem with minimal interaction between nephrin and c-Abl. Moreover, Ang II promoted c-Abl phosphorylation and interaction between c-Abl and SH2 domain–containing 5′-inositol phosphatase 2 (SHIP2). c-Abl small interfering RNA (siRNA) and STI571 (c-Abl inhibitor) provided protection against Ang II–induced podocyte injury, suppressed the Ang II-induced c-Abl–SHIP2 interaction and SHIP2 phosphorylation, and maintained a stable level of nephrin phosphorylation. These results indicate that c-Abl is a molecular chaperone of nephrin signaling and the SHIP2-Akt pathway and that the released c-Abl contributes to Ang II–induced podocyte injury. PMID:26510503

  10. Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

    Directory of Open Access Journals (Sweden)

    Hawdon John M

    2009-04-01

    Full Text Available Abstract Background Third-stage infective larvae (L3 of hookworms are in an obligatory state of developmental arrest that ends upon entering the definitive host, where they receive a signal that re-activates development. Recovery from the developmentally arrested dauer stage of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development. Results To determine if hookworm 14-3-3 proteins play a similar role in L3 activation, hookworm FTT-2 was identified and tested for its ability to interact with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-FTT-2, and was most closely related to FTT-2 from other nematodes. Ac-FTT-2 was expressed in HEK 293T cells, and was recognized by an antibody against human 14-3-3β isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-DAF-16 when co-expressed in serum-stimulated HEK 293T cells. This interaction requires intact Akt consensus phosphorylation sites at serine107 and threonine312, but not serine381. Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated L3 or adult A. caninum. Conclusion The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

  11. Knockdown of Akt Sensitizes Osteosarcoma Cells to Apoptosis Induced by Cisplatin Treatment

    Directory of Open Access Journals (Sweden)

    Changwei Yang

    2011-05-01

    Full Text Available Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin. Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA. It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation. The results of this study suggest that knockdown of Akt2 expression may have therapeutic applications in enhancing the efficacy of chemotherapy in patients with osteosarcoma.

  12. Knockdown of Akt Sensitizes Osteosarcoma Cells to Apoptosis Induced by Cisplatin Treatment

    Science.gov (United States)

    Zhang, Guoyou; Li, Ming; Zhu, Xiaodong; Bai, Yushu; Yang, Changwei

    2011-01-01

    Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin. Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA). It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation. The results of this study suggest that knockdown of Akt2 expression may have therapeutic applications in enhancing the efficacy of chemotherapy in patients with osteosarcoma. PMID:21686164

  13. 20-Hydroxyeicosatetraenoic acid impairs endothelial insulin signaling by inducing phosphorylation of the insulin receptor substrate-1 at Ser616.

    Directory of Open Access Journals (Sweden)

    Xuguang Li

    Full Text Available 20-Hydroxyeicosatetraenoic acid (20-HETE induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.

  14. Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

    International Nuclear Information System (INIS)

    Highlights: → In 3T3-L1 adipocytes iAs3+ decreases insulin-stimulated glucose uptake. → iAs3+ attenuates insulin-induced phosphorylation of AKT S473. → iAs3+ activates the cellular adaptive oxidative stress response. → iAs3+ impairs insulin-stimulated ROS signaling. → iAs3+ decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 μM) inorganic arsenite (iAs3+) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs3+ exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs3+ exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes

  15. Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Peng [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); School of Public Health, China Medical University, Shenyang 110001 (China); Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Sun, Guifan [School of Public Health, China Medical University, Shenyang 110001 (China); Andersen, Melvin E. [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)

    2011-04-08

    Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4

  16. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    DEFF Research Database (Denmark)

    Adelfinger, L; Turecek, R; Ivankova, K; Jensen, Anders A.; Moss, S.J.; Gassmann, M; Bettler, B

    2014-01-01

    reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G...... regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted...

  17. Resistance exercise, but not endurance exercise, induces IKKβ phosphorylation in human skeletal muscle of training-accustomed individuals

    DEFF Research Database (Denmark)

    Møller, Andreas Buch; Vendelbo, Mikkel Holm; Rahbek, Stine Klejs; Clasen, Berthil Forrest; Schjerling, Peter; Vissing, Kristian; Jessen, Niels

    2013-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) is considered an important role in the muscular adaptations to exercise. It has been proposed that exercise-induced signaling to mTORC1 do not require classic growth factor PI3K/Akt signaling. Activation of IKKβ and the mitogen-activated protein......), TSC1, MAPK, and upstream Akt activators, along with gene expression of selected cytokines, in skeletal muscles from these subjects. Biopsies were sampled prior to, immediately after, and in the recovery period following resistance exercise, endurance exercise, and control interventions. The major...... repeated biopsy sampling on protein expression and phosphorylation was observed. In conclusion, resistance exercise, but not endurance exercise, increases IKKβ phosphorylation in trained human subjects, which support the idea that IKKβ can influence the activation of mTORC1 in human skeletal muscle....

  18. Isorhynchophylline Protects PC12 Cells Against Beta-Amyloid-Induced Apoptosis via PI3K/Akt Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yan-Fang Xian

    2013-01-01

    Full Text Available The neurotoxicity of amyloid-β (Aβ has been implicated as a critical cause of Alzheimer’s disease. Isorhynchophylline (IRN, an oxindole alkaloid isolated from Uncaria rhynchophylla, exerts neuroprotective effect against Aβ25–35-induced neurotoxicity in vitro. However, the exact mechanism for its neuroprotective effect is not well understood. The present study aimed to investigate the molecular mechanisms underlying the protective action of IRN against Aβ25–35-induced neurotoxicity in cultured rat pheochromocytoma (PC12 cells. Pretreatment with IRN significantly increased the cell viability, inhibited the release of lactate dehydrogenase and the extent of DNA fragmentation in Aβ25–35-treated cells. IRN treatment was able to enhance the protein levels of phosphorylated Akt (p-Akt and glycogen synthase kinase-3β (p-GSK-3β. Lithium chloride blocked Aβ25–35-induced cellular apoptosis in a similar manner as IRN, suggesting that GSK-3β inhibition was involved in neuroprotective action of IRN. Pretreatment with LY294002 completely abolished the protective effects of IRN. Furthermore, IRN reversed Aβ25–35-induced attenuation in the level of phosphorylated cyclic AMP response element binding protein (p-CREB and the effect of IRN could be blocked by the PI3K inhibitor. These experimental findings unambiguously suggested that the protective effect of IRN against Aβ25–35-induced apoptosis in PC12 cells was associated with the enhancement of p-CREB expression via PI3K/Akt/GSK-3β signaling pathway.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. DMA mitigates ionizing radiation induced damage in Balb/c mice through Akt/NFκB/PTEN pathway

    International Nuclear Information System (INIS)

    Ionizing radiation is associated with massive apoptosis in tumor as well as in radiosensitive organs. DMA, (5-(4-methylpiperazin-1-yl)-2-(2'-(3,4-dimethoxy-phenyl)-5'-benzimidazolyl) a cytoprotective radiomodulator, work in dual mode of action as free radical quencher and modifier of genomic instability caused by radiation. We observed 34% radioprotection with 50 mg/Kg bw intravenous dose of DMA in Balb/c mice at 8 Gy. DMA treatment before irradiation restored the normal crypts and villi architecture in Balb/c mice. The villi height was restored equivalent to control group in DMA treated animals, whereas, it was degenerated in irradiated animals. IR-induced apoptosis was reduced in spleen in presence of DMA as a result of preservation of splenic lymphocytes from radiation. This clearly exhibits the radioprotective ability of DMA to mitigate radiation induced tissue damage. IR-induced S phase check point was overcome by DMA. DMA promoted activation and phosphorylation of GSK3β through the activation of Akt in Balb/c mice. There was reduction in PTEN level in DMA pretreated mice where as it was upregulated in irradiated mice. Relative enhanced kinase activity of Akt was observed in DMA treated Balb/c mice and irradiated A549, MRC5 cell lines. There was no significant radioprotection in DMA treated Akt siRNA transfected cells in comparison to only Akt siRNA transfected cells with increasing dose of radiation. Akt activation was found in a dose-dependent manner by DMA through Luciferase reporter assay. We observed that DMA treated HEK cells transfected with control siRNA, resulted in less early apoptotic cells within 24h, but radiation (5 Gy) treated cells showed 20% early apoptotic cells within 3 h which were reduced to 12% at 3 h, 9% at 6 h and 8% at 24 h in DMA+radiation treated cells determined by Annexin V binding assay. Further molecular mRNA expression analysis of key regulatory genes unveil that DMA inhibited p21 and augmented Akt and Gadd45 in

  2. Suppression of inflammatory responses during MOG-induced experimental autoimmune encephalomyelitis is regulated by AKT3 signaling

    OpenAIRE

    Tsiperson, Vladislav; Gruber, Ross C; Goldberg, Michael; Jordan, Ayana; Weinger, Jason G.; Macian, Fernando; Shafit-Zagardo, Bridget

    2013-01-01

    AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biological processes. AKT3 is expressed in immune cells, and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hyp...

  3. Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

    Science.gov (United States)

    Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

    2016-09-01

    Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy. PMID:26328540

  4. Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

    Science.gov (United States)

    Degani, CH.; Kawatsuji, M.; Halmann, M.

    1975-01-01

    It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

  5. Celastrol prevents cadmium-induced neuronal cell death via targeting JNK and PTEN-Akt/mTOR network.

    Science.gov (United States)

    Chen, Sujuan; Gu, Chenjian; Xu, Chong; Zhang, Jinfei; Xu, Yijiao; Ren, Qian; Guo, Min; Huang, Shile; Chen, Long

    2014-01-01

    Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Celastrol, a plant-derived triterpene, has shown neuroprotective effects in various disease models. However, little is known regarding the effect of celastrol on Cd-induced neurotoxicity. Here, we show that celastrol protected against Cd-induced apoptotic cell death in neuronal cells. This is supported by the findings that celastrol strikingly attenuated Cd-induced viability reduction, morphological change, nuclear fragmentation, and condensation, as well as activation of caspase-3 in neuronal cells. Concurrently, celastrol remarkably blocked Cd-induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinases 1/2 and p38, in neuronal cells. Inhibition of JNK by SP600125 or over-expression of dominant negative c-Jun potentiated celastrol protection against Cd-induced cell death. Furthermore, pre-treatment with celastrol prevented Cd down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in neuronal cells. Over-expression of wild-type PTEN enhanced celastrol inhibition of Cd-activated Akt/mTOR signaling and cell death in neuronal cells. The findings indicate that celastrol prevents Cd-induced neuronal cell death via targeting JNK and PTEN-Akt/mTOR network. Our results strongly suggest that celastrol may be exploited for the prevention of Cd-induced neurodegenerative disorders. Celastrol, a plant-derived triterpene, has shown neuroprotective effects. However, little is known regarding the effect of celastrol on cadmium (Cd) neurotoxicity. This study underscores that celastrol prevents Cd-induced neuronal apoptosis via inhibiting activation of JNK (c-Jun N-terminal kinase) and Akt/mTOR network. Celastrol suppresses Cd-activated Akt/mTOR pathway by elevating PTEN (phosphatase and tensin homolog). The

  6. Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

    International Nuclear Information System (INIS)

    Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) index in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo-IR in

  7. Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying [Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016 (China); Wang, Jianwei, E-mail: wangjianwei1968@gmail.com [Department of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016 (China); Gu, Tieguang [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences, Sydney, NSW 2000 Australia (Australia); Yamahara, Johji [Pharmafood Institute, Kyoto 602-8136 (Japan); Li, Yuhao, E-mail: yuhao@sitcm.edu.au [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences, Sydney, NSW 2000 Australia (Australia)

    2014-06-01

    Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) index in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo-IR in

  8. Carnosic acid induces apoptosis associated with mitochondrial dysfunction and Akt inactivation in HepG2 cells.

    Science.gov (United States)

    Xiang, Qisen; Ma, Yunfang; Dong, Jilin; Shen, Ruiling

    2015-02-01

    Carnosic acid (CA), a phenolic diterpene isolated from rosemary, shows potential benefits in health promotion and disease prevention. In the present study, the cytotoxic and apoptotic-inducing effects of CA on human hepatocellular carcinoma HepG2 cells were investigated. The MTT assay results indicated that CA decreased cell viability in HepG2 cells in a dose-dependent manner. Treatment with CA caused a rapid Caspase-3 activation and subsequently proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), both of which were markers of cells undergoing apoptosis. CA also dissipated mitochondrial membrane potential and decreased the ratio of Bcl-2/Bax protein, which mediated cytosolic translocation of cytochrome c from the mitochondria. Furthermore, CA reduced the phosphorylation of Akt, which was partially inhibited by insulin, an activator of phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway. In conclusion, our data suggest that the mitochondrial dysfunction and deactivation of Akt may contribute to the apoptosis-inducing effects of CA. PMID:25265205

  9. Phosphorylation of Akt by SC79 Prevents Iron Accumulation and Ameliorates Early Brain Injury in a Model of Experimental Subarachnoid Hemorrhage

    Directory of Open Access Journals (Sweden)

    Shuangying Hao

    2016-03-01

    Full Text Available Previous studies have demonstrated that activation of Akt may alleviate early brain injury (EBI following subarachnoid hemorrhage (SAH. This study is undertaken to determine whether iron metabolism is involved in the beneficial effect of Akt activation after SAH. Therefore, we used a novel molecule, SC79, to activate Akt in an experimental Sprague–Dawley rat model of SAH. Rats were randomly divided into four groups as follows: sham, SAH, SAH + vehicle, SAH + SC79. The results confirmed that SC79 effectively enhanced the defense against oxidative stress and alleviated EBI in the temporal lobe after SAH. Interestingly, we found that phosphorylation of Akt by SC79 reduced cell surface transferrin receptor-mediated iron uptake and promoted ferroportin-mediated iron transport after SAH. As a result, SC79 administration diminished the iron content in the brain tissue. Moreover, the impaired Fe-S cluster biogenesis was recovered and loss of the activities of the Fe-S cluster-containing enzymes were regained, indicating that injured mitochondrial functions are restored to healthy levels. These findings suggest that disrupted iron homeostasis could contribute to EBI and Akt activation may regulate iron metabolism to relieve iron toxicity, further protecting neurons from EBI after SAH.

  10. Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt.

    Science.gov (United States)

    Chen, Jialin; Chen, Peng; Backman, Ludvig J; Zhou, Qingjun; Danielson, Patrik

    2016-01-01

    The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway. PMID:27174608

  11. VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-κB signaling pathway

    Directory of Open Access Journals (Sweden)

    Huang Chuanshu

    2006-03-01

    Full Text Available Abstract Background Hypoxia-induced mitogenic factor (HIMF is developmentally regulated and plays an important role in lung pathogenesis. We initially found that HIMF promotes vascular tubule formation in a matrigel plug model. In this study, we investigated the mechanisms which HIMF enhances expression of vascular endothelial growth factor (VEGF in lung tissues and epithelial cells. Methods Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, VEGF expression was examined by immunohistochemical staining and Western blot. The promoter-luciferase reporter assay, RT-PCR, and Western blot were performed to examine the effects of HIMF on VEGF expression in mouse lung epithelial cell line MLE-12. The activation of NF-kappa B (NF-κB and phosphorylation of Akt, IKK and IκBα were examined by luciferase assay and Western blot, respectively. Results Intratracheal instillation of HIMF protein resulted in significant increase of VEGF, mainly localized to airway epithelial and alveolar type II cells. Deletion of NF-κB binding sites within VEGF promoter abolished HIMF-induced VEGF expression in MLE-12 cells, suggesting that activation of NF-κB is essential for VEGF upregulation induced by HIMF. Stimulation of lung epithelial cells by HIMF resulted in phosphorylation of IKK and IκBα, leading to activation of NF-κB. In addition, HIMF strongly induced Akt phosphorylation, and suppression of Akt activation by specific inhibitors and dominant negative mutants for PI-3K, and IKK or IκBα blocked HIMF-induced NF-κB activation and attenuated HIMF-induced VEGF production. Conclusion These results suggest that HIMF enhances VEGF production in mouse lung epithelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis.

  12. Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Murai Atsuko

    2012-07-01

    Full Text Available Abstract Background Canine hemangiosarcoma (HSA is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines. Results Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs, that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1 at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1 at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines. Conclusions Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in

  13. AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines

    Directory of Open Access Journals (Sweden)

    Leal P

    2013-10-01

    Full Text Available Pamela Leal,1,* Patricia Garcia,2,* Alejandra Sandoval,1 Kurt Buchegger,1 Helga Weber,1 Oscar Tapia,1 Juan C Roa1,2 1Department of Pathology, Universidad de La Frontera, Center of Genetical and Immunological Studies-Scientific and Technological Bioresource Nucleus, Temuco, 2Department of Pathology, Pontificia Universidad Catolica de Chile, Santiago, Chile *These authors contributed equally to this paperBackground: Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines.Methods: Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression.Results: Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18, early cancer (84.6%, 22/26, and advanced cancer (88.3%, 121/137. No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall

  14. Astaxanthin reduces isoflurane-induced neuroapoptosis via the PI3K/Akt pathway.

    Science.gov (United States)

    Wang, Chun-Mei; Cai, Xiao-Lan; Wen, Qing-Ping

    2016-05-01

    Astaxanthin is an oxygen-containing derivative of carotenoids that effectively suppresses reactive oxygen and has nutritional and medicinal value. The mechanisms underlying the effects of astaxanthin on isoflurane‑induced neuroapoptosis remain to be fully understood. The present study was conducted to evaluate the protective effect of astaxanthin to reduce isoflurane‑induced neuroapoptosis and to investigate the underlying mechanisms. The results demonstrated that isoflurane induced brain damage, increased caspase‑3 activity and suppressed the phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) signaling pathway in an in vivo model. However, treatment with astaxanthin significantly inhibited brain damage, suppressed caspase‑3 activity and upregulated the PI3K/Akt pathway in the isoflurane‑induced rats. Furthermore, isoflurane suppressed cell growth, induced cell apoptosis, enhanced caspase‑3 activity and downregulated the PI3K/Akt pathway in organotypic hippocampal slice culture. Administration of astaxanthin significantly promoted cell growth, reduced cell apoptosis and caspase‑3 activity, and upregulated the PI3K/Akt pathway and isoflurane‑induced neuroapoptosis. The present study demonstrated that downregulation of the PI3K/Akt pathway reduced the effect of astaxanthin to protect against isoflurane‑induced neuroapoptosis in the in vitro model. The results of the current study suggested that the protective effect of astaxanthin reduces the isoflurane-induced neuroapoptosis via activation of the PI3K/Akt signaling pathway. PMID:27035665

  15. TG-interacting factor transcriptionally induced by AKT/FOXO3A is a negative regulator that antagonizes arsenic trioxide-induced cancer cell apoptosis

    International Nuclear Information System (INIS)

    Arsenic trioxide (ATO) is a multi-target drug approved by the Food and Drug Administration as the first-line chemotherapeutic agent for the treatment of acute promyelocytic leukemia. In addition, several clinical trials are being conducted with arsenic-based drugs for the treatment of other hematological malignancies and solid tumors. However, ATO's modest clinical efficacy on some cancers, and potential toxic effects on humans have been reported. Determining how best to reduce these adverse effects while increasing its therapeutic efficacy is obviously a critical issue. Previously, we demonstrated that the JNK-induced complex formation of phosphorylated c-Jun and TG-interacting factor (TGIF) antagonizes ERK-induced cyclin-dependent kinase inhibitor CDKN1A (p21WAF1/CIP1) expression and resultant apoptosis in response to ATO in A431 cells. Surprisingly, at low-concentrations (0.1–0.2 μM), ATO increased cellular proliferation, migration and invasion, involving TGIF expression, however, at high-concentrations (5–20 μM), ATO induced cell apoptosis. Using a promoter analysis, TGIF was transcriptionally regulated by ATO at the FOXO3A binding site (− 1486 to − 1479 bp) via the c-Src/EGFR/AKT pathway. Stable overexpression of TGIF promoted advancing the cell cycle into the S phase, and attenuated 20 μM ATO-induced apoptosis. Furthermore, blockage of the AKT pathway enhanced ATO-induced CDKN1A expression and resultant apoptosis in cancer cells, but overexpression of AKT1 inhibited CDKN1A expression. Therefore, we suggest that TGIF is transcriptionally regulated by the c-Src/EGFR/AKT pathway, which plays a role as a negative regulator in antagonizing ATO-induced CDKN1A expression and resultant apoptosis. Suppression of these antagonistic effects might be a promising therapeutic strategy toward improving clinical efficacy of ATO. - Highlights: • ATO-induced biphasic survival responses of cancer cells depend on low- or high-concentrations. • TGIF mediates

  16. TG-interacting factor transcriptionally induced by AKT/FOXO3A is a negative regulator that antagonizes arsenic trioxide-induced cancer cell apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zi-Miao; Tseng, Hong-Yu; Cheng, Ya-Ling [Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China); Yeh, Bi-Wen [Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China); Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Wu, Wen-Jeng [Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Huang, Huei-Sheng, E-mail: huanghs@mail.ncku.edu.tw [Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China)

    2015-05-15

    Arsenic trioxide (ATO) is a multi-target drug approved by the Food and Drug Administration as the first-line chemotherapeutic agent for the treatment of acute promyelocytic leukemia. In addition, several clinical trials are being conducted with arsenic-based drugs for the treatment of other hematological malignancies and solid tumors. However, ATO's modest clinical efficacy on some cancers, and potential toxic effects on humans have been reported. Determining how best to reduce these adverse effects while increasing its therapeutic efficacy is obviously a critical issue. Previously, we demonstrated that the JNK-induced complex formation of phosphorylated c-Jun and TG-interacting factor (TGIF) antagonizes ERK-induced cyclin-dependent kinase inhibitor CDKN1A (p21{sup WAF1/CIP1}) expression and resultant apoptosis in response to ATO in A431 cells. Surprisingly, at low-concentrations (0.1–0.2 μM), ATO increased cellular proliferation, migration and invasion, involving TGIF expression, however, at high-concentrations (5–20 μM), ATO induced cell apoptosis. Using a promoter analysis, TGIF was transcriptionally regulated by ATO at the FOXO3A binding site (− 1486 to − 1479 bp) via the c-Src/EGFR/AKT pathway. Stable overexpression of TGIF promoted advancing the cell cycle into the S phase, and attenuated 20 μM ATO-induced apoptosis. Furthermore, blockage of the AKT pathway enhanced ATO-induced CDKN1A expression and resultant apoptosis in cancer cells, but overexpression of AKT1 inhibited CDKN1A expression. Therefore, we suggest that TGIF is transcriptionally regulated by the c-Src/EGFR/AKT pathway, which plays a role as a negative regulator in antagonizing ATO-induced CDKN1A expression and resultant apoptosis. Suppression of these antagonistic effects might be a promising therapeutic strategy toward improving clinical efficacy of ATO. - Highlights: • ATO-induced biphasic survival responses of cancer cells depend on low- or high-concentrations. • TGIF

  17. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    International Nuclear Information System (INIS)

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT

  18. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xian-Ying; Chen, Wei [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Fan, Jun-Ting [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Song, Ran; Wang, Lu [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Gu, Yan-Hong [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Zeng, Guang-Zhi [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Shen, Yan; Wu, Xue-Feng [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Tan, Ning-Hua, E-mail: nhtan@mail.kib.ac.cn [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Xu, Qiang, E-mail: molpharm@163.com [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Sun, Yang, E-mail: yangsun@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China)

    2013-02-15

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT.

  19. Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Park, Eun-Seok [Department of Applied Biochemistry, Division of Life Science, College of Health and Biomedical Science, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Kang, Shin-il [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of); Yoo, Kyu-dong [Hazardous Substances Analysis Division, Gwangju Regional Food and Drug Administration, Gwangju (Korea, Republic of); Lee, Mi-Yea [Department of Nursing Kyungbok University, Pocheon (Korea, Republic of); Yoo, Hwan-Soo; Hong, Jin-Tae [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of); Shin, Hwa-Sup [Department of Applied Biochemistry, Division of Life Science, College of Health and Biomedical Science, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk (Korea, Republic of); Yun, Yeo-Pyo, E-mail: ypyun@chungbuk.ac.kr [College of Pharmacy Medical Research Center, Chungbuk National University, Cheongju (Korea, Republic of)

    2013-04-15

    The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway. - Highlights: ► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway.

  20. Luteolin Inhibits Angiotensin II-Stimulated VSMC Proliferation and Migration through Downregulation of Akt Phosphorylation

    OpenAIRE

    Tongda Xu; Hong Zhu; Dongye Li; Yasong Lang; Lijuan Cao; Yang Liu; Wanling Wu; Dan Chen

    2015-01-01

    Luteolin is a naturally occurring flavonoid found in many plants that possesses cardioprotective properties. The purpose of this study was to elucidate the effect of luteolin on vascular smooth muscle cells (VSMCs) proliferation and migration induced by Angiotensin II (Ang II) and to investigate the mechanism(s) of action of this compound. Rat VSMCs were cultured in vitro, and the proliferation and migration of these cells following Ang II stimulation were monitored. Different doses of luteol...

  1. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro

    OpenAIRE

    Ji Young Park; Mei Hong; Qi Jia; Young-Chul Lee; Taddesse Yayeh; Eujin Hyun; Dong-Mi Kwak; Jae Youl Cho; Man Hee Rhee

    2012-01-01

    Pistacia chinensis (Chinese pistache) is a widely grown plant in southern China where the galls extract is a common practice in folk medicine. However, extracts from this plant have never been attempted for their cardiovascular protective effects in experimental setting. Here therefore we aimed to investigate the antiplatelet activity of Pistacia chinensis methanolic extract (PCME) in ADP stimulated rat platelets in vitro. PCME (2.5–20 μg/mL) inhibited ADP-induced platelet aggregation. While ...

  2. Phenylbutyric acid induces the cellular senescence through an Akt/p21WAF1 signaling pathway

    International Nuclear Information System (INIS)

    Highlights: ► Phenylbutyric acid induces cellular senescence. ► Phenylbutyric acid activates Akt kinase. ► The knockdown of PERK also can induce cellular senescence. ► Akt/p21WAF1 pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins – PERK, ATF6 and IRE1 – initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21WAF1 induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21WAF1 pathway by PERK inhibition.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Isoorientin induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Li; Wang, Jing; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo, E-mail: xueboliu@yahoo.com.cn

    2012-11-15

    Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer. Highlights:

  5. Isoorientin induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells

    International Nuclear Information System (INIS)

    Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer. Highlights:

  6. Over-expression of NYGGF4 inhibits glucose transport in 3T3-L1 adipocytes via attenuated phosphorylation of IRS-1 and Akt

    Institute of Scientific and Technical Information of China (English)

    Chun-mei ZHANG; Xiao-hui CHEN; Bin WANG; Feng LIU; Xia CHI; Mei-ling TONG; Yu-hui NI; Rong-hua CHEN; Xi-rong GUO

    2009-01-01

    Aim: NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese patients. The purpose of this study was to investigate the effects of NYGGF4 on basal and insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes and to understand the underlying mechanisms. Methods: 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. Glucose uptake was determined by measuring 2-deoxy-D-[3H]glucose uptake into the adipocytes. Immunoblotting was performed to detect the translocation of insulin-sensitive glu-cose transporter 4 (GLUT4). Immunoblotting also was used to measure the phosphorylation and total protein contents of insulin signaling proteins such as the insulin receptor (IR), insulin receptor substrate (IRS)-I, Akt, ERK1/2, p38, and JNK. Results: NYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phos-phorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, and JNK. Conclusion: NYGGF4 regulates the functions of IRS-1 and Akt, decreases GLUT4 translocation and reduces glucose uptake in response to insulin. These observations highlight the potential role of NYGGF4 in glucose homeostasis and possibly in the pathogenesis of obesity.

  7. Arsenite-induced stress signaling: Modulation of the phosphoinositide 3′-kinase/Akt/FoxO signaling cascade

    Directory of Open Access Journals (Sweden)

    Ingrit Hamann

    2013-01-01

    Full Text Available FoxO transcription factors and their regulators in the phosphoinositide 3′-kinase (PI3K/Akt signaling pathway play an important role in the control of cellular processes involved in carcinogenesis, such as proliferation and apoptosis. We have previously demonstrated that physiologically relevant heavy metal ions, such as copper or zinc ions, can stimulate this pathway, triggering phosphorylation and nuclear export of FoxO transcription factors. The present study aims at investigating the effect of arsenite on FoxO transcription factors and the role of PI3K/Akt signaling therein. Exposure of HaCaT human keratinocytes to arsenite resulted in a distinct decrease of glutathione levels only at cytotoxic concentrations. In contrast, a strong phosphorylation of FoxO1a/FoxO3a and Akt was observed at subcytotoxic concentrations of arsenite in HaCaT human keratinocytes. A time- and concentration-dependent increase in phosphorylation of FoxO1a and FoxO3a at sites known to be phosphorylated by Akt as well as phosphorylation of Akt at Ser-473 was detected. These phosphorylations were blunted in the presence of wortmannin, pointing to the involvement of PI3K.

  8. Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

    Directory of Open Access Journals (Sweden)

    Dongdong Zhang

    2015-01-01

    Full Text Available Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer’s disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ25–35 induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer’s disease.

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

    Directory of Open Access Journals (Sweden)

    Yüksel Korkmaz

    2010-01-01

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

  10. Role of SIRT1-mediated mitochondrial and Akt pathways in glioblastoma cell death induced by Cotinus coggygria flavonoid nanoliposomes

    Directory of Open Access Journals (Sweden)

    Wang G

    2015-08-01

    phosphorylated p53. Together, these results indicated SIRT1/p53-mediated cell death was induced by CCF-NLs, but not by extracellular signal-regulated kinase, in DBTRG-05MG cells. Overall, this study suggested caspase-dependent activation of both the intrinsic and extrinsic signaling pathways, probably through blockade of the SIRT1/p53-mediated mitochondrial and Akt pathways to exert the proapoptotic effect of CCF-NLs in DBTRG-05MG GBM cells. Keywords: Cotinus coggygria flavonoid nanoliposomes, cell death, SIRT1, mitochondrial, PI3K/Akt pathway

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

    Directory of Open Access Journals (Sweden)

    Deng Wang

    2012-03-01

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

  12. Puerarin protects rat kidney from lead-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chan-Min, E-mail: lcm9009@126.com [School of Life Science, Xuzhou Normal University, No.101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Xuzhou City, Jiangsu Province (China); Ma, Jie-Qiong [School of Chemical Engineering, China University of Mining and Technology, Xuzhou 221008, Xuzhou City, Jiangsu Province (China); Sun, Yun-Zhi [School of Life Science, Xuzhou Normal University, No.101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Xuzhou City, Jiangsu Province (China)

    2012-02-01

    Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. However, its protective effects against lead (Pb) induced injury in kidney have not been clarified. The aim of the present study was to investigate the effects of puerarin on renal oxidative stress and apoptosis in rats exposed to Pb. Wistar rats were exposed to lead acetate in the drinking water (500 mg Pb/l) with or without puerarin co-administration (100, 200, 300 and 400 mg PU/kg intragastrically once daily) for 75 days. Our data showed that puerarin significantly prevented Pb-induced nephrotoxicity in a dose-dependent manner, indicated by both diagnostic indicators of kidney damage (serum urea, uric acid and creatinine) and histopathological analysis. Moreover, Pb-induced profound elevation of reactive oxygen species (ROS) production and oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of intracellular reduced glutathione (GSH) level in kidney, were suppressed by treatment with puerarin. Furthermore, TUNEL assay showed that Pb-induced apoptosis in rat kidney was significantly inhibited by puerarin. In exploring the underlying mechanisms of puerarin action, we found that activities of caspase-3 were markedly inhibited by the treatment of puerarin in the kidney of Pb-treated rats. Puerarin increased phosphorylated Akt, phosphorylated eNOS and NO levels in kidney, which in turn inactivated pro-apoptotic signaling events including inhibition of mitochondria cytochrome c release and restoration of the balance between pro- and anti-apoptotic Bcl-2 proteins in kidney of Pb-treated rats. In conclusion, these results suggested that the inhibition of Pb-induced apoptosis by puerarin is due at least in part to its antioxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway. Highlights: ► Puerarin prevented lead-induced nephrototoxicity. ► Puerarin reduced lead-induced increase in ROS and TBARS production

  13. PI3K/Akt/FoxO3a signaling mediates cardioprotection of FGF-2 against hydrogen peroxide-induced apoptosis in H9c2 cells.

    Science.gov (United States)

    Liu, Mi-Hua; Li, Guo-Hua; Peng, Li-Jun; Qu, Shun-Lin; Zhang, Yuan; Peng, Juan; Luo, Xin-Yuan; Hu, Heng-Jing; Ren, Zhong; Liu, Yao; Tang, Hui; Liu, Lu-Shan; Tang, Zhi-Han; Jiang, Zhi-Sheng

    2016-03-01

    Cardiovascular disease is a growing major global public health problem. Oxidative stress is regarded as one of the key regulators of pathological physiology, which eventually leads to cardiovascular disease. However, mechanisms by which FGF-2 rescues cells from oxidative stress damage in cardiovascular disease is not fully elucidated. Herein this study was designed to investigate the protective effects of FGF-2 in H2O2-induced apoptosis of H9c2 cardiomyocytes, as well as the possible signaling pathway involved. Apoptosis of H9c2 cardiomyocytes was induced by H2O2 and assessed using methyl thiazolyl tetrazolium assay, Hoechst, and TUNEL staining. Cells were pretreated with PI3K/Akt inhibitor LY294002 to investigate the possible PI3K/Akt pathways involved in the protection of FGF-2. The levels of p-Akt, p-FoxO3a, and Bim were detected by immunoblotting. Stimulation with H2O2 decreased the phosphorylation of Akt and FoxO3a, and induced nuclear localization of FoxO3a and apoptosis of H9c2 cells. These effects of H2O2 were abrogated by pretreatment with FGF-2. Furthermore, the protective effects of FGF-2 were abolished by PI3K/Akt inhibitor LY294002. In conclusion, our data suggest that FGF-2 protects against H2O2-induced apoptosis of H9c2 cardiomyocytes via activation of the PI3K/Akt/FoxO3a pathway. PMID:26899709

  14. Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt.

    Science.gov (United States)

    Deng, Li-Juan; Hu, Li-Ping; Peng, Qun-Long; Yang, Xiao-Lin; Bai, Liang-Liang; Yiu, Anita; Li, Yong; Tian, Hai-Yan; Ye, Wen-Cai; Zhang, Dong-Mei

    2014-08-01

    Hellebrigenin, one of bufadienolides belonging to cardioactive steroids, was found in skin secretions of toads and plants of Helleborus and Kalanchoe genera. In searching for natural constituents with anti-hepatoma activities, we found that hellebrigenin, isolated from traditional Chinese medicine Venenum Bufonis, potently reduced the viability and colony formation of human hepatocellular carcinoma cells HepG2, and went on to explore the underlying molecular mechanisms. Our results demonstrated that hellebrigenin triggered DNA damage through DNA double-stranded breaks and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-CDK1 (Tyr(15)) and Cyclin B1, and down-regulation of p-CDC25C (Ser(216)). It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP. In addition, Akt expression and phosphorylation were inhibited by hellebrigenin, whereas Akt silencing with siRNA significantly blocked cell cycle arrest but enhanced apoptosis induced by hellebrigenin. Activation of Akt by human insulin-like growth factor I (hIGF-I) could obviously attenuate hellebrigenin-induced cell death. In summary, our study is the first to report the efficacy of hellebrigenin against HepG2 and elucidated its molecular mechanisms including DNA damage, mitochondria collapse, cell cycle arrest and apoptosis, which will contribute to the development of hellebrigenin into a chemotherapeutic agent in the treatment of liver cancer. PMID:24954031

  15. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    International Nuclear Information System (INIS)

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

  16. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Xiao [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Whitten, Douglas A. [Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Wu, Ming [Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Chan, Christina [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Wilkerson, Curtis G. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Pestka, James J., E-mail: pestka@msu.edu [Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824 (United States)

    2013-04-15

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

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

    Science.gov (United States)

    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

  18. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    Science.gov (United States)

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  19. The role of the Akt/mTOR pathway in tobacco-carcinogen induced lung tumorigenesis

    OpenAIRE

    Memmott, Regan M.; Dennis, Phillip A.

    2009-01-01

    Lung cancer is the leading cause of cancer-related death in the United States, and 85–90% of lung cancer cases are associated with tobacco use. Tobacco components promote lung tumorigenesis through genotoxic effects, as well as through biochemical modulation of signaling pathways such as the Akt/mTOR pathway that regulate cell proliferation and survival. This review will describe cell surface receptors and other upstream components required for tobacco-carcinogen induced activation of Akt and...

  20. UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

    International Nuclear Information System (INIS)

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2-/- mouse embryonic fibroblasts (MEFs) while Akt1-/- MEFs show cell cycle arrest. Here, we find that Akt1-/- MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated β-galactosidase (SA β-gal) staining indicate that Akt1-/- MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1-/- MEFs suppressed SA β-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1-/- MEFs, suggesting that UV light induces premature senescence in Akt1-/- MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

  1. mTOR Promotes Survival and Astrocytic Characteristics Induced by Pten/Akt Signaling in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Xiaoyi Hu

    2005-04-01

    Full Text Available 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.

  2. PIP3 but not PIP2 increases GLUT4 surface expression and glucose metabolism mediated by AKT/PKCζ/λ phosphorylation in 3T3L1 adipocytes

    OpenAIRE

    Manna, Prasenjit; Jain, Sushil K.

    2013-01-01

    PIP3 (phosphatidylinositol-3,4,5-triphosphate) and PIP2 (phosphatidylinositol-4,5-biphosphate) are two well-known membrane bound polyphosphoinositides. Diabetes is associated with impaired glucose metabolism. Using a 3T3L1 adipocyte cell model, this study investigated the roles of PIP3 and PIP2 on insulin stimulated glucose metabolism in high glucose (HG) treated cells. Exogenous PIP3 supplementation (1, 5, or 10 nM) increased the phosphorylation of AKT and PKCζ/λ, which in turn upregulated G...

  3. Platycodin-D Induced Autophagy in Non-Small Cell Lung Cancer Cells via PI3K/Akt/mTOR and MAPK Signaling Pathways.

    Science.gov (United States)

    Zhao, Ruolin; Chen, Meijuan; Jiang, Zequn; Zhao, Fengming; Xi, Beili; Zhang, Xu; Fu, Haian; Zhou, Kunfu

    2015-01-01

    Platycodin-D (PD) is an effective triterpene saponin extracted from the root of Platycodon grandiflorum which has been used clinically to treat pulmonary diseases in traditional Chinese medicine. Recently, it has been reported that PD has anti-tumor effects in various cancer models through the induction of apoptosis. However, whether PD induces autophagy in both cell lines and its molecular mechanisms have not been elucidated. Here, our present study confirmed that PD induced autophagy in both NCI-H460 and A549 cells via up-regulating the expression levels of Atg-3, Atg-7 and Beclin-1. Meanwhile, PD contributed to the up-regulation of LC3-II at both protein and mRNA levels. Further detection of the PI3K/Akt/mTOR signaling pathway compared to LY294002 (PI3K kinase inhibitor), RAP (mTOR kinase inhibitor) and insulin (an activator of PI3K/Akt/mTOR signaling pathway) showed that PD induced autophagy through inhibiting the pathway at p-Akt (Ser473), p-p70S6K (Thr389) and p-4EBP1 (Thr37/46) in both cell lines. Moreover, the examination of MAPK signaling pathway showed that PD treatment increased the phosphorylation of JNK and p38 MAPK, while decreased the phosphorylation of Erk1/2 in both cell lines. Additionally, the effects assessed with a panel of pharmacologic inhibitors, including U0126 (Erk1/2 kinase inhibitor), SP600125 (JNK kinase inhibitor) and SB203580 (p38 MAPK kinase inhibitor) suggested that the activation of JNK and p38 MAPK participated in PD-induced autophagy. Taken together, these findings suggested that PD induced autophagy in NCI-H460 and A549 cells through inhibiting PI3K/Akt/mTOR signaling pathway and activating JNK and p38 MAPK signaling pathways. Therefore, PD may be an alternative compound for NSCLC therapy. PMID:26078792

  4. SCD1 Expression is dispensable for hepatocarcinogenesis induced by AKT and Ras oncogenes in mice.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC, de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA into monounsaturated fatty acids (MUFA, is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1(-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.

  5. Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment

    DEFF Research Database (Denmark)

    Højlund, Kurt; Glintborg, Dorte; Andersen, Nicoline Resen;

    2008-01-01

    OBJECTIVE: Insulin resistance in skeletal muscle is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the molecular mechanisms underlying skeletal muscle insulin resistance and the insulin-sensitizing effect of thiazolidinediones in PCOS in vivo are...... less well characterized. RESEARCH DESIGN AND METHODS: We determined molecular mediators of insulin signaling to glucose transport in skeletal muscle biopsies of 24 PCOS patients and 14 matched control subjects metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry......, and we examined the effect of 16 weeks of treatment with pioglitazone in PCOS patients. RESULTS: Impaired insulin-mediated total (R(d)) oxidative and nonoxidative glucose disposal (NOGD) was paralleled by reduced insulin-stimulated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation in...

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

    Science.gov (United States)

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

    2015-04-15

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

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

    Science.gov (United States)

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

    2016-05-01

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

  8. Altered Regulation of Contraction-Induced Akt/mTOR/p70S6k Pathway Signaling in Skeletal Muscle of the Obese Zucker Rat

    Directory of Open Access Journals (Sweden)

    Anjaiah Katta

    2009-01-01

    Full Text Available Increased muscle loading results in the phosphorylation of the 70 kDa ribosomal S6 kinase (p70S6k, and this event is strongly correlated with the degree of muscle adaptation following resistance exercise. Whether insulin resistance or the comorbidities associated with this disorder may affect the ability of skeletal muscle to activate p70S6k signaling following an exercise stimulus remains unclear. Here, we compare the contraction-induced activation of p70S6k signaling in the plantaris muscles of lean and insulin resistant obese Zucker rats following a single bout of increased contractile loading. Compared to lean animals, the basal phosphorylation of p70S6k (Thr389; 37.2% and Thr421/Ser424; 101.4%, Akt (Thr308; 25.1%, and mTOR (Ser2448; 63.0% was higher in obese animals. Contraction increased the phosphorylation of p70S6k (Thr389, Akt (Ser473, and mTOR (Ser2448 in both models however the magnitude and kinetics of activation differed between models. These results suggest that contraction-induced activation of p70S6k signaling is altered in the muscle of the insulin resistant obese Zucker rat.

  9. Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

    Directory of Open Access Journals (Sweden)

    Hunter Randy

    2008-01-01

    Full Text Available Abstract Background Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS; however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1 the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS-induced iNOS activity and nitric oxide (NO generation by microglia; (2 the differential role of p38 mitogen-activated protein kinase (p38 MAPK, c-Jun NH(2-terminal kinase (JNK, and phosphoinositide 3-kinase (PI3K on LPS-induced NO generation; and (3 the regulation of p38 MAPK, JNK, and PI3K by pioglitazone. Methods Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH-positive neurons were counted in mesencephalic neuron-microglia mixed cultures. Results Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of

  10. Nitric oxide synthase and breast cancer: role of TIMP-1 in NO-mediated Akt activation.

    Directory of Open Access Journals (Sweden)

    Lisa A Ridnour

    Full Text Available 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.

  11. Thymosin β4 induces invasion and migration of human colorectal cancer cells through the ILK/AKT/β-catenin signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Piao, Zhengri [Research Center for Molecular Therapeutic to GI Tract of Cancer Center, Chonnam National University Hwasun Hospital, Hwasun (Korea, Republic of); Center for Creative Biomedical Scientists (BK-21 Plus Project), Chonnam National University Medical School, Gwangju (Korea, Republic of); Hong, Chang-Soo [Research Center for Molecular Therapeutic to GI Tract of Cancer Center, Chonnam National University Hwasun Hospital, Hwasun (Korea, Republic of); Jung, Mi-Ran [Department of Gastroenterologic Surgery, Chonnam National University Hwasun Hospital, Hwasun (Korea, Republic of); Choi, Chan [Department of Pathology, Chonnam National University Hwasun Hospital, Hwasun (Korea, Republic of); Park, Young-Kyu, E-mail: parkyk@jnu.ac.kr [Research Center for Molecular Therapeutic to GI Tract of Cancer Center, Chonnam National University Hwasun Hospital, Hwasun (Korea, Republic of); Center for Creative Biomedical Scientists (BK-21 Plus Project), Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Gastroenterologic Surgery, Chonnam National University Hwasun Hospital, Hwasun (Korea, Republic of)

    2014-09-26

    Highlights: • Tβ4 is overexpressed in human colorectal cancer cells. • The overexpression of Tβ4 is correlated with stage of colorectal cancer. • Tβ4 stimulates cell adhesion, invasion, migration and EMT. • Tβ4 activates the ILK/AKT/β-catenin signaling pathway. - Abstract: Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.

  12. Thymosin β4 induces invasion and migration of human colorectal cancer cells through the ILK/AKT/β-catenin signaling pathway

    International Nuclear Information System (INIS)

    Highlights: • Tβ4 is overexpressed in human colorectal cancer cells. • The overexpression of Tβ4 is correlated with stage of colorectal cancer. • Tβ4 stimulates cell adhesion, invasion, migration and EMT. • Tβ4 activates the ILK/AKT/β-catenin signaling pathway. - Abstract: Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment

  13. Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 in human skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Pehmøller, Christian; Kristensen, Jonas Møller;

    2014-01-01

    We investigated the phosphorylation signatures of two Rab GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers...... TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK was regulating...... phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring...

  14. Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways.

    Science.gov (United States)

    Bin, Geng; Bo, Zhang; Jing, Wang; Jin, Jiang; Xiaoyi, Tan; Cong, Chen; Liping, An; Jinglin, Ma; Cuifang, Wang; Yonggang, Chen; Yayi, Xia

    2016-05-01

    TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. PMID:27060196

  15. Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells.

    Science.gov (United States)

    Kawiak, Anna; Lojkowska, Ewa

    2016-01-01

    The phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer. PMID:26840401

  16. Ramentaceone, a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Anna Kawiak

    Full Text Available The phosphoinositide 3-kinase (PI3K signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer.

  17. Protection against MPP(+)-induced neurotoxicity in SH-SY5Y cells by tormentic acid via the activation of PI3-K/Akt/GSK3β pathway.

    Science.gov (United States)

    Zhao, Qing; Ye, Junli; Wei, Na; Fong, Chichun; Dong, Xiaoli

    2016-07-01

    The cause of Parkinson's disease (PD) could be ascribed to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta, and thus molecules with neuroprotective ability may have therapeutic value against PD. In the current study, the neuroprotective effects and underlying mechanisms of tormentic acid (TA), a naturally occurring triterpene extracted from medicinal plants such as Rosa rugosa and Potentilla chinensis, were evaluated in a widely used cellular PD model in which neurotoxicity was induced by MPP(+) in cultured SH-SY5Y cells. We found that TA at 1-30 μM substantially protected against MPP(+)-induced neurotoxicity, as evidenced by the increase in cell viability, decrease in lactate dehydrogenase release and the reduction in apoptotic nuclei. Moreover, TA effectively inhibited the elevated intracellular accumulation of reactive oxygen species as well as Bax/Bcl-2 ratio caused by MPP(+). Most importantly, TA markedly reversed the inhibition of protein expression of phosphorylated Akt (Ser 473) and phosphorylated GSK3β (Ser 9) caused by MPP(+). LY294002, the specific inhibitor of PI3-K, significantly abrogated the up-regulated phosphorylated Akt and phosphorylated GSK3β offered by TA, suggesting that the neuroprotection of TA was mainly dependent on the activation of PI3-K/Akt/GSK3β signaling pathway. The results taken together indicate that TA may be a potential candidate for further preclinical study aimed at the prevention and treatment of PD. PMID:26994872

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

    Science.gov (United States)

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

    2008-02-28

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

  19. MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma

    International Nuclear Information System (INIS)

    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

  20. MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuqin [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Zheng, Lin [Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong Province (China); Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Ding, Yi [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Li, Qi [Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Wang, Rong [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Liu, Tongxin; Sun, Quanquan [Department of Radiation Oncology, Cancer Hospital, Hangzhou, Zhejiang Province (China); Yang, Hua [Department of Radiation Oncology, Nanhai Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Peng, Shunli [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Wang, Wei, E-mail: wangwei9500@hotmail.com [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China); Chen, Longhua, E-mail: chenlhsmu@126.com [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province (China)

    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.

  1. Ellagic acid protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway

    International Nuclear Information System (INIS)

    Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized low-density lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. Previous studies have shown that the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase/nitric oxide (PI3K/Akt/eNOS/NO) pathway is involved in oxLDL-induced endothelial apoptosis. Ellagic acid, a natural polyphenol found in berries and nuts, has in recent years been the subject of intense research within the fields of cancer and inflammation. However, its protective effects against oxLDL-induced injury in vascular endothelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. Our results showed that pretreatment with ellagic acid (5-20 μM) significantly attenuated oxLDL-induced cytotoxicity, apoptotic features, and generation of reactive oxygen species (ROS). In addition, the anti-apoptotic effect of ellagic acid was partially inhibited by a PI3K inhibitor (wortmannin) and a specific eNOS inhibitor (cavtratin) but not by an ERK inhibitor (PD98059). In exploring the underlying mechanisms of ellagic acid action, we found that oxLDL decreased Akt and eNOS phosphorylation, which in turn activated NF-κB and downstream pro-apoptotic signaling events including calcium accumulation, destabilization of mitochondrial permeability, and disruption of the balance between pro- and anti-apoptotic Bcl-2 proteins. Those alterations induced by oxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of oxLDL-induced endothelial apoptosis by ellagic acid is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

  2. Concomitant activation of the PI3K/Akt and ERK1/2 signalling is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells.

    Science.gov (United States)

    Yin, Jian; Hao, Zhichao; Ma, Yuanyuan; Liao, Shuang; Li, Xianxian; Fu, Jing; Wu, Yeke; Shen, Jiefei; Zhang, Ping; Li, Xiaoyu; Wang, Hang

    2014-05-01

    IL-6 has a dual role in bone remodelling. The ERK1/2 pathway partially upregulated IL-6 secretion in osteocyte-like MLO-Y4 cells exposed to CCF. We have now investigated the possible role of phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway in the CCF-induced IL-6 expression. MLO-Y4 cells were treated with CCF 2,000 µstrain, 2 Hz, or 10, 30 min, 1, 3 and 6 h. IL-6 expression, Akt and ERK1/2 and PI3K/Akt phosphorylation were determined by RT-PCR, ELISA and Western blotting. Inhibition of PI3K/Akt with LY294002 or ERK1/2 with PD98059 significantly attenuated IL-6 upregulation, and IL-6 expression was abolished by inhibiting both pathways. Inhibition of one pathway downregulated the other's phosphorylation level. In conclusion, concomitant activation of PI3K/Akt and ERK1/2 pathways mediated IL-6 expression in MLO-Y4 cells under CCF. PMID:24375569

  3. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  4. Embelin-Induced Apoptosis of Human Prostate Cancer Cells Is Mediated through Modulation of Akt and β-Catenin Signaling.

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    Nahee Park

    Full Text Available There is increasing evidence that embelin, an active component of Embelia ribes, induces apoptosis in human cancer cells, but the detailed mechanisms are still unclear. Here, we have investigated the effect of embelin on the growth of human prostate cancer cells. Embelin strongly inhibited cell growth especially in human prostate cancer cell lines, including PC3, DU145, LNCaP-LN3 and normal prostate epithelial cell, RWPE-1 compared to breast cancer (MDA-MB-231, MCF-7, and T47D, hepatoma (HepG2, Hep3B, and HuH-7, or choriocarcinoma (JEG-3. We observed that embelin induced apoptosis of PC3 cells in a time-dependent manner correlated with decreased expression of Bcl-2, Bcl-xL, and Mcl-1, increased translocation of Bax into mitochondria, and a reduction in the mitochondrial membrane potential. Furthermore, embelin induced voltage-dependent anion channel (VDAC 1 expression and oligomerization, which may promote cytochrome c and AIF release. Because embelin was able to inhibit Akt activation and cyclooxygenase-2 expression, the effects on Wnt/ β-catenin signaling were determined. Embelin activated glycogen synthase kinase (GSK-3β by preventing phosphorylation and suppressed β-catenin expression. Attenuation of β-catenin-mediated TCF transcriptional activity and gene transcription, such as cyclin D1, c-myc, and matrix metalloproteinase (MMP-7, were shown in embelin-treated cells. The changes in β-catenin levels in response to embelin were blocked by lithium chloride, a GSK-3 inhibitor, indicating that embelin may decrease β-catenin expression via GSK-3β activation. Furthermore, exposure of PC3 cells to embelin resulted in a significant decrease in cell migration and invasion. In conclusion, these findings suggest that inhibition of Akt signaling and activation of GSK-3β partially contributes to the pro-apoptotic effect of embelin in prostate cancer cells.

  5. Adriamycin induces H2AX phosphorylation in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    Zhong-Xiang Li; Ting-Ting Wang; Yan-Ting Wu; Chen-Ming Xu; Min-Yue Dong; Jian-Zhong Sheng; He-Feng Huang

    2008-01-01

    Aim: To investigate whether adriamycin induces DNA damage and the formation of γH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa. Methods: Human spermatozoa were treated with adriamycin at different concentrations. γH2AX was analyzed by immunofluorescent staining and flow cytometry and double- strand breaks (DSB) were detected by the comet assay. Results: The neutral comet assay revealed that the treatment with adriamycin at 2 μg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 μg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of γH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with γH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with γH2AX. Conclusion: Human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.

  6. Salinomycin induces cell death and differentiation in head and neck squamous cell carcinoma stem cells despite activation of epithelial-mesenchymal transition and Akt

    International Nuclear Information System (INIS)

    Cancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells. MTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose. In putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR. These results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and

  7. Naringenin-induced migration of embrynoic trophectoderm cells is mediated via PI3K/AKT and ERK1/2 MAPK signaling cascades.

    Science.gov (United States)

    Lim, Whasun; Song, Gwonhwa

    2016-06-15

    For successful pregnancy, a well-coordinated network of growth factors, nutrients and hormones is required for fetal-maternal interactions. Naringenin, as a weak phytoestrogen, improves diabetes, inflammation, neuronal diseases, cardiovascular diseases and cancers. However, the role of naringenin in migration mechanism(s) of peri-implantation conceptuses is unknown. Therefore, in the present study, we determined the effects of naringenin on migration of porcine trophectoderm (pTr) cells, which is a known in vitro model for research on trophectoderm cell biology and placental-fetal developmental biology, in order to assess intracellular signal transduction pathways activated by naringenin. Migration of pTr cells increased in a dose-dependent manner in response to naringenin. Also, naringenin activated the phosphorylation of AKT and ERK1/2 proteins in a dose-dependent manner and those proteins were abundant mainly in the cytoplasm of naringenin-treated pTr cells. Within 30 min after treatment with 20 μM naringenin, the abundance of phosphorylated EKR1/2, P70S6K, P90RSK and S6K proteins increased, and then returned to basal levels by 120 min whereas the abundance of AKT increased gradually to 120 min post-treatment. However, the phosphorylation of AKT, P70S6K, P90RSK and S6K was reduced in naringenin-induced pTr cells pre-treated with a PI3K inhibitor (LY294002). Also, a MEK1/2 inhibitor (U0126) significantly decreased naringenin-induced phosphorylation of ERK1/2, P70S6K and S6K proteins in pTr cells. Moreover, the naringenin-stimulated migration of pTr cells was suppressed by LY294002 and U0126. Collectively, results of the present study suggest that naringenin supports migration of pTr cells through PI3K/AKT and ERK1/2 MAPK signaling pathways crucial for orchestrating conceptus-uterine interactions. PMID:26994515

  8. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    Science.gov (United States)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

  9. Saponins from the roots of Platycodon grandiflorum suppresses TGFβ1-induced epithelial-mesenchymal transition via repression of PI3K/Akt, ERK1/2 and Smad2/3 pathway in human lung carcinoma A549 cells.

    Science.gov (United States)

    Choi, Jae Ho; Hwang, Yong Pil; Kim, Hyung Gyun; Khanal, Tilak; Do, Minh Truong; Jin, Sun Woo; Han, Hwa Jeong; Lee, Hyun Sun; Lee, Young Chun; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-01-01

    Transforming growth factor β (TGFβ) is a multifunctional cytokine that induces growth arrest, tissue fibrosis, and epithelial-mesenchymal transition (EMT) through activation of Smad and non-Smad signaling pathways. EMT is the differentiation switch by which polarized epithelial cells differentiate into contractile and motile mesenchymal cells. Our previous studies have shown that saponins from the roots of Platycodon grandiflorum (CKS) have antiinflammatory, antioxidant, antimetastatic, and hepatoprotective effects. In this study, we investigated the inhibitory effect of CKS on TGFβ1-induced alterations characteristic of EMT in human lung carcinoma A549 cells. We found that CKS-treated cells displayed inhibited TGFβ1-mediated E-cadherin downregulation and Vimentin upregulation and also retained epithelial morphology. Furthermore, TGFβ1-increased Snail expression, a repressor of E-cadherin and an inducer of the EMT, was reduced by CKS. CKS inhibited TGFβ1-induced phosphorylation of Akt, ERK1/2, and glycogen synthase kinase-3β (GSK-3β). Inhibition of PI3K/Akt and ERK1/2 also blocked TGFβ1-induced GSK-3β phosphorylation and Snail activation. Furthermore, TGFβ1-increased Snail expression was reduced by selective inhibitors of Akt and ERK1/2. Moreover, CKS treatment attenuated TGFβ1-induced Smad2/3 phosphorylation and upregulated Smad7 expression. These results indicate that pretreatment with the CKS inhibits the TGFβ1-induced EMT through PI3K/Akt, ERK1/2, GSK-3β and Smad2/3 in human lung carcinoma cells. PMID:24341702

  10. NVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3K/AKT/ mTOR phosphorylation

    Directory of Open Access Journals (Sweden)

    Sun ZH

    2015-01-01

    Full Text Available Zhihua Sun,2,* Qiuhui li,1,* Sheng Zhang,1 Jing Chen,1 Lili Huang,3 Jinghua Ren,1 Yu Chang,1 Yichen Liang,1 Gang Wu1 1Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 2Oncology department, Xiangyang central Hospital, Xiangyang, Hubei, People’s Republic of China; 3Radiation Oncology Department, Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong, People's Republic of China *These authors contributed equally to this work Background: Patients harboring activating mutations in epidermal growth factor receptors (EGFR are particularly sensitive to EGFR tyrosine kinase inhibitors (TKIs. However, most patients develop an acquired resistance after a period of about 10 months. This study focuses on the therapeutic effect of NVP-BEZ235, a dual inhibitor of phosphatidylinositol- 3-kinase/mammalian target of rapamycin (PI3K/mTOR, in gefitinib-resistant non-small cell lung cancer. Methods: H1975 cell line was validated as a gefitinib-resistant cell model by the nucleotide-sequence analysis. We used the 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay to detect the growth of H1975 cell line in vitro. H1975 cells' migration was detected by the migration assay. Xenograft models were used to investigate the growth of gefitinib-resistant non-small cell lung cancer in vivo. Western blot and immunohistochemical analysis were used to investigate the level of PI3K/protein kinase B(AKT/mTOR signaling pathway proteins. Results: We show that NVP-BEZ235 effectively inhibited the growth of H1975 cells in vivo as well as in vitro. Similarly, H1975 cell migration was reduced by NVP-BEZ235. Further experiments revealed that NVP-BEZ235 attenuated the phosphorylation of PI3K/AKT/mTOR signaling pathway proteins. Conclusion: Taken together, we suggest that NVP-BEZ235 inhibits gefitinib-resistant tumor growth by downregulating PI3K/AKT

  11. The Flavonoid Apigenin Ameliorates Cisplatin-Induced Nephrotoxicity through Reduction of p53 Activation and Promotion of PI3K/Akt Pathway in Human Renal Proximal Tubular Epithelial Cells

    Science.gov (United States)

    Ju, Sung Min; Kang, Jun Gue; Bae, Jun Sang; Pae, Hyun Ock; Lyu, Yeoung Su; Jeon, Byung Hun

    2015-01-01

    Apigenin is a member of the flavone subclass of flavonoids present in fruits and vegetables. Apigenin has long been considered to have various biological activities, such as antioxidant, anti-inflammatory, and antitumorigenic properties, in various cell types. Cisplatin was known to exhibit cytotoxic effect to renal cells by inducing apoptosis through activation of p53. The present study investigated the antiapoptotic effects of apigenin on the cisplatin-treated human renal proximal tubular epithelial (HK-2) cells. HK-2 cells were pretreated with apigenin (5, 10, 20 μM) for 1 h and then treated with 40 μM cisplatin for various times. Apigenin inhibited the cisplatin-induced apoptosis of HK-2 cells. Interestingly, apigenin itself exerted cytostatic activity because of its ability to induce cell cycle arrest. Apigenin inhibited caspase-3 activity and PARP cleavage in cisplatin-treated cells. Apigenin reduced cisplatin-induced phosphorylation and expression of p53, with no significant influence on production of ROS that is known to induce p53 activation. Furthermore, apigenin promoted cisplatin-induced Akt phosphorylation, suggesting that enhanced Akt activation may be involved in cytoprotection. Taken together, these results suggest that apigenin ameliorates cisplatin-induced apoptosis through reduction of p53 activation and promotion of PI3K/Akt pathway in HK-2 cells. PMID:26089934

  12. GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons

    Directory of Open Access Journals (Sweden)

    Sohn Sunghyang

    2010-06-01

    Full Text Available Abstract Background Gangliosides, sialic acid-containing glycosphingolipids exist in mammalian cell membranes particularly neuronal membranes. The trisialoganglioside (GT1b is one of the major brain gangliosides and acts as an endogenous regulator in the brain. We previously showed GT1b induces mesencephalic dopaminergic (DA neuronal death, both in vivo and in vitro. We further investigate the underlying mechanisms of GT1b neurotoxicity. Results Consistent with earlier findings, GT1b attenuated the DA neuron number and dopamine uptake level in mesencephalic cultures. Morphological evidence revealed GT1b-induced chromatin condensation and nuclear fragmentation as well as an increased number of TUNEL-positive cells, compared to control cultures. Interestingly, while GT1b enhanced caspase-3 activity, DEVD, a caspase-3 inhibitor, failed to rescue DA neuronal death. Immunoblot analysis revealed that GT1b inactivates Akt through dephosphorylation at both Ser473 and Thr308, subsequent dephosphorylation of GSK-3β, a substrate of Akt, and hyperphosphorylation of tau, downstream of GSK-3β. Moreover, a GSK-3β specific inhibitor, L803-mt, attenuated tau phosphorylation and rescued DA neurons from cell death in mesencephalic cultures. Conclusion Our data provide novel evidence that a Akt/GSK-3β/tau-dependent, but not caspase-3 signaling pathway plays a pivotal role in GT1b-mediated neurotoxic actions on mesencephalic DA neurons.

  13. Contribution of CFTR to Alveolar Fluid Clearance by Lipoxin A4 via PI3K/Akt Pathway in LPS-Induced Acute Lung Injury

    Directory of Open Access Journals (Sweden)

    Yi Yang

    2013-01-01

    Full Text Available The lipoxins are the first proresolution mediators to be recognized and described as the endogenous “braking signals” for inflammation. We evaluated the anti-inflammatory and proresolution bioactions of lipoxin A4 in our lipopolysaccharide (LPS-induced lung injury model. We demonstrated that lipoxin A4 significantly improved histology of rat lungs and inhibited IL-6 and TNF-α in LPS-induced lung injury. In addition, lipoxin A4 increased alveolar fluid clearance (AFC and the effect of lipoxin A4 on AFC was abolished by CFTRinh-172 (a specific inhibitor of CFTR. Moreover, lipoxin A4 could increase cystic fibrosis transmembrane conductance regulator (CFTR protein expression in vitro and in vivo. In rat primary alveolar type II (ATII cells, LPS decreased CFTR protein expression via activation of PI3K/Akt, and lipoxin A4 suppressed LPS-stimulated phosphorylation of Akt. These results showed that lipoxin A4 enhanced CFTR protein expression and increased AFC via PI3K/Akt pathway. Thus, lipoxin A4 may provide a potential therapeutic approach for acute lung injury.

  14. Nitidine chloride inhibits proliferation, induces apoptosis via the Akt pathway and exhibits a synergistic effect with doxorubicin in ovarian cancer cells.

    Science.gov (United States)

    Ding, Feng; Liu, Tianfeng; Yu, Nina; Li, Shihong; Zhang, Xiaofei; Zheng, Guanghong; Lv, Chunming; Mou, Kai; Xu, Jia; Li, Bo; Wang, Surong; Song, Haibo

    2016-09-01

    Nitidine chloride (NC) exhibits anti-tumor properties in various types of tumor. However, to the best of our knowledge there is no previous evidence of NC involvement in the apoptosis or proliferation of ovarian cancer cells and the underlying molecular mechanisms. The present study aimed to investigate the influence of NC on the viability and apoptosis of ovarian cancer cells and the synergistic effect NC and doxorubicin (DOX) may have on ovarian cancer cells. The viability and proliferation of ovarian cancer cells were examined using a methyl thiazolyl tetrazolium assay and 3H-thymidine incorporation assay. The apoptotic rate of ovarian cancer cells was detected by flow cytometry. The expression of apoptosis‑associated proteins and Akt serine/threonine kinase 1 (Akt) were determined by western blot analysis following NC treatment. The inhibitory effect of NC on the proliferation of ovarian cancer cells was demonstrated in a time and dose‑dependent manner. The pro-apoptotic effect of NC on ovarian cancer cells was also observed. It was determined that NC significantly downregulated the protein expression levels of B‑cell CLL/lymphoma 2 (Bcl-2) and upregulated the expression of Bcl‑2‑associated X protein, p53, caspase‑3 and ‑9. NC suppressed Akt phosphorylation. Additionally, the present study demonstrated that the effect of NC on the proliferation and apoptosis of ovarian cancer cells was Akt‑dependent by using the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt signaling pathway inhibitor, LY294002. NC exhibited a synergistic inhibitory effect on the viability of ovarian cancer cells when combined with DOX. The current study demonstrated that NC inhibited the proliferation and induced the apoptosis of ovarian cancer cells via the Akt signaling pathway and highlighted its potential clinical application for the treatment of ovarian cancer. PMID:27485415

  15. Quercetin ameliorates ischemia/reperfusion-induced cognitive deficits by inhibiting ASK1/JNK3/caspase-3 by enhancing the Akt signaling pathway.

    Science.gov (United States)

    Pei, Bing; Yang, Miaomiao; Qi, Xiaoyan; Shen, Xin; Chen, Xing; Zhang, Fayong

    2016-09-01

    Cerebral ischemia/reperfusion (I/R) is a major cause of severe disability and death all worldwide. However, therapeutic options to minimize the detrimental effects of cerebral I/R injury are limited. Recent research has demonstrated that quercetin mediates neuroprotective effects associated with the activation of the Akt signaling pathway in the cerebral I/R brain. Therefore, the aim of this study was to further investigate the mechanisms of cognitive deficits induced by cerebral I/R injury and the effects of quercetin on these mechanisms. First, we assessed anxiety-like behavioral and cognitive impairment using the open field test and the Morris water maze test, respectively. Next, we examined the severity of apoptosis by staining hippocampal neurons by the Cresyl violet method. Third, we used western blot analysis to investigate the expression of total and phosphorylated Akt, ASK1, JNK3, c-Jun and caspase-3 after I/R injury. Our results revealed that mice subjected to bilateral common carotid occlusion exhibited severe anxiety-like behavior, learning and memory impairment, cell damage and apoptosis. These severe effects were attenuated by administration of quercetin. Further, western blot analysis revealed that quercetin increased p-Akt expression and decreased p-ASK1, p-JNK3 and cleaved caspase-3 expression after cerebral I/R injury and led to inhibition of neuronal apoptosis. Conversely, treatment with LY294002 (a selective inhibitor of Akt1) reversed the effects of quercetin. In conclusion, these findings highlight the important role of quercetin in protecting against cognitive deficits and inhibiting neuronal apoptosis via the Akt signaling pathway. We believe that quercetin might prove to be a useful therapeutic component in treating cerebral I/R diseases in the near future. PMID:27450812

  16. Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production.

    Directory of Open Access Journals (Sweden)

    Tai-An Chen

    Full Text Available BACKGROUND: The application of viral elements in tumor therapy is one facet of cancer research. Recombinant capsid protein VP1 (rVP1 of foot-and-mouth disease virus has previously been demonstrated to induce apoptosis in cancer cell lines. Here, we aim to further investigate its apoptotic mechanism and possible anti-metastatic effect in murine models of hepatocellular carcinoma (HCC, one of the most common human cancers worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with rVP1 inhibited cell proliferation in two murine HCC cell lines, BNL and Hepa1-6, with IC₅₀ values in the range of 0.1-0.2 µM. rVP1 also induced apoptosis in these cells, which was mediated by Akt deactivation and dissociation of Ku70-Bax, and resulted in conformational changes and mitochondrial translocation of Bax, leading to the activation of caspases-9, -3 and -7. Treatment with 0.025 µM rVP1, which did not affect the viability of normal hepatocytes, suppressed cell migration and invasion via attenuating CCL2 production. The production of CCL2 was modulated by Akt-dependent NF-κB activation that was decreased after rVP1 treatment. The in vivo antitumor effects of rVP1 were assessed in both subcutaneous and orthotopic mouse models of HCC in immune-competent BALB/c mice. Intratumoral delivery of rVP1 inhibited subcutaneous tumor growth as a result of increased apoptosis. Intravenous administration of rVP1 in an orthotopic HCC model suppressed tumor growth, inhibited intra-hepatic metastasis, and prolonged survival. Furthermore, a decrease in the serum level of CCL2 was observed in rVP1-treated mice. CONCLUSIONS/SIGNIFICANCE: The data presented herein suggest that, via inhibiting Akt phosphorylation, rVP1 suppresses the growth, migration, and invasion of murine HCC cells by inducing apoptosis and attenuating CCL2 production both in vitro and in vivo. Recombinant protein VP1 thus has the potential to be developed as a new therapeutic agent for HCC.

  17. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation

    International Nuclear Information System (INIS)

    Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three

  18. Effects of streptozotocin-induced type 1 maternal diabetes on PI3K/AKT signaling pathway in the hippocampus of rat neonates.

    Science.gov (United States)

    Hami, Javad; Kerachian, Mohammad-Amin; Karimi, Razieh; Haghir, Hossein; Sadr-Nabavi, Ariane

    2016-06-01

    Diabetes in pregnancy impairs hippocampus development in offspring, leading to behavioral problems and learning deficits. Phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway plays a pivotal role in the regulation of neuronal proliferation, survival and death. The present study was designed to examine the effects of maternal diabetes on PKB/Akt expression and phosphorylation in the developing rat hippocampus. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at first postnatal day (P1). The hippocampal expression and phosphorylation level of PKB/Akt, one of the key molecules in PI3K/AKT signaling pathway, was evaluated using real-time polymerase chain reaction (PCR) and western blot analysis. We found a significant bilateral downregulation of AKT1 gene expression in the hippocampus of pups born to diabetic mothers (p < 0.05). Interestingly, our results revealed a marked upregulation of Akt1 gene in insulin-treated group compared with other groups (p < 0.05). The western blot analysis also showed the reduction of phosphorylation level of all AKT isoforms in both diabetic and insulin-treated groups compared with control (p < 0.05). Moreover, the results showed a significant increase in phosphorylation level of AKT in insulin-treated group compared with the diabetic group. These results represent that diabetes during pregnancy strongly influences the regulation of PKB/AKT in the developing rat hippocampus. Furthermore, although the control of glycemia by insulin administration is not sufficient to prevent the alterations in PKB/Akt expression, it modulates the phosphorylation process, thus ultimately resulting in a situation comparable to that found in the normal condition. PMID:26479041

  19. AKT1E17K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer

    Science.gov (United States)

    Malanga, Donatella; Belmonte, Stefania; Colelli, Fabiana; Scarfò, Marzia; De Marco, Carmela; Oliveira, Duarte Mendes; Mirante, Teresa; Camastra, Caterina; Gagliardi, Monica; Rizzuto, Antonia; Mignogna, Chiara; Paciello, Orlando; Papparella, Serenella; Fagman, Henrik; Viglietto, Giuseppe

    2016-01-01

    The hotspot AKT1E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. Recently, we have demonstrated that AKT1E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1E17K mice) we demonstrate that AKT1E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype. PMID:26859676

  20. Calycosin ameliorates diabetes-induced cognitive impairments in rats by reducing oxidative stress via the PI3K/Akt/GSK-3β signaling pathway.

    Science.gov (United States)

    Wang, Xiang; Zhao, Linhui

    2016-04-29

    Diabetic encephalopathy is one of the most prevalent chronic complications of diabetes mellitus (DM), but there is currently no effective method of prevention nor proven therapeutic regimen for it. In this study, we investigated the effects of calycosin on cognitive behavior and the potential mechanism involved in streptozocin-induced diabetic rats. The effects of diabetes and calycosin treatment on spatial learning and memory were evaluated using the Morris Water Maze, passive avoidance and motor coordination tests. Histological analysis of the hippocampus cornu ammonis 1 (CA1) region was conducted in rats. The decreased expression of the synapsin (SYN) and postsynatptic density protein (PSD-95), as well as brain-derived neurotrophic factor (BDNF) in diabetic rats was measured by quantitative real-time PCR and western blot. Treatment with calycosin promoted a reduction in the expression of SYN, PSD-95 and BDNF. In addition, diabetic rats showed increased MDA levels, and decreased SOD levels and GSH-Px activities in the hippocampus, as well as increased AChE activity in the cerebral cortex; these changes were reversed by calycosin supplementation. Thus, the impairment of learning and memory in STZ-induced diabetic rats was alleviated by calycosin, and that the degree of alleviation was associated with oxidative stress. We also found that calycosin treatment significantly stimulated Akt phosphorylation and decreased GSK-3β and tau phosphorylation, and that these changes could be restored by the PI3K/Akt inhibitor LY294002. In conclusion, calycosin had a beneficial effect on the amelioration, prevention and treatment of diabetes-associated cognitive deficits, through its involvement in oxidative stress, synaptic function and the PI3K/Akt/GSK-3β pathway. PMID:26970304

  1. Orexin A-mediated AKT signaling in the dentate gyrus contributes to the acquisition, expression and reinstatement of morphine-induced conditioned place preference.

    Science.gov (United States)

    Guo, Sui-Jun; Cui, Yu; Huang, Zhen-Zhen; Liu, Huan; Zhang, Xue-Qin; Jiang, Jin-Xiang; Xin, Wen-Jun

    2016-05-01

    Accumulating evidence indicates that the hippocampal dentate gyrus (DG), a critical brain region contributing to learning and memory, is involved in the addiction and relapse to abused drugs. Emerging studies also suggest the role of orexin signaling in the rewarding behavior induced by repeated exposure to opiates. In the present study, we investigated the dynamic adaptation of orexin signaling in the DG and its functional significance in the acquisition, expression, maintenance of and relapse to rewarding behavior induced by morphine. Repeated place conditioning with morphine significantly increased the orexin A content released from the lateral hypothalamic area projecting neurons into the DG. Local infusions of orexin A into the DG sensitized the acquisition of and relapse to the conditioned place preference induced by morphine. The application of the orexin receptor type 1 (OXR1) antagonist SB334867 significantly abolished the acquisition, expression and maintenance of the conditioned place preference induced by repeated exposure to morphine. Furthermore, the significant increase of the phosphorylation of AKT in the DG was associated with preference for the morphine-paired chamber in rats, which was reversed by the local administration of an OXR1 antagonist. Thus, these findings suggested that the dynamic upregulation of orexin A signaling, via the AKT pathway in the DG, may promote the acquisition and maintenance of opioid-induced craving behaviors and may increase sensitivity to the rewarding effect of subsequent opioids. PMID:25757577

  2. Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yun-Jeong; Cho, Hyun-Ji [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Magae, Junji [Magae Bioscience Institute, 49-4 Fujimidai, Tsukuba 300-1263 (Japan); Lee, In-Kyu [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Park, Keun-Gyu, E-mail: kpark@knu.ac.kr [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

    2013-12-15

    Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.

  3. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hag Dong [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Jang, Chang-Young [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Choe, Jeong Min [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Kim, Joon, E-mail: joonkim@korea.ac.kr [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  4. RLIP76-dependent suppression of PI3K/AKT/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jing; Song, Qi; Cai, Yi; Wang, Peng; Wang, Min; Zhang, Dong, E-mail: zhangd1117@yahoo.com

    2015-08-07

    MicroRNA-101 (miR-101) participates in carcinogenesis and tumor progression in various cancers. However, its biological functions in prostate cancer are still unclear. Here, we demonstrate that miR-101 represents a critical role in regulating cell apoptosis in prostate cancer cells. We first demonstrated that miR-101 treatment promoted apoptosis in DU145 and PC3 cells by using flow cytometric analysis and transmission electron microscopy (TEM). To verify the mechanisms, we identified a novel miR-101 target, Ral binding protein 1 (RLIP76). We found miR-101 transfection significantly suppresses RLIP76 expression, which can transactivate phosphorylation of PI3K-Akt signaling, and resulted in an amplification of Bcl2-induced apoptosis. Furthermore, we demonstrated that RLIP76 overexpression could reverse the anti-tumor effects of miR-101 in DU145 and PC3 cells by using flow cytometry assay and MTT assay. Taken together, our results revealed that the effect of miR-101 on prostate cancer cell apoptosis was due to RLIP76 regulation of the PI3K/Akt/Bcl-2 signaling pathway. - Highlights: • miR-101 inhibited prostate cancer cell proliferation and enhanced apoptosis. • miR-101 directly targeted and regulated RLIP76 expression. • miR-101 suppressed PI3K/Akt/Bcl-2 signaling pathway by targeting RLIP76.

  5. Fucoidan from Undaria pinnatifida prevents vascular dysfunction through PI3K/Akt/eNOS-dependent mechanisms in the l-NAME-induced hypertensive rat model.

    Science.gov (United States)

    Li, Xiaofei; Li, Jian; Li, Zhike; Sang, Ying; Niu, Yunhui; Zhang, Qianying; Ding, Hong; Yin, Shanye

    2016-05-18

    Despite major scientific advances in its prevention, treatment and care, hypertension remains a serious condition that might lead to long-term complications such as heart disease and stroke. The great majority of forms of hypertension eventually result from an increased vasomotor tone activity that is regulated by endothelial NOS (eNOS) in vascular endothelium. Here, we examined the effect of fucoidan on eNOS activation in human umbilical vein endothelial cells (HUVECs). We also examined the effects of functional components of Undaria pinnatifida fucoidan on blood pressure and vascular function in eNOS inhibition-induced hypertensive rats in vivo. Our results suggest that fucoidan increased nitric oxide production by activating eNOS and Akt phosphorylation, which could be impaired by Akt or eNOS inhibitors. In the hypertensive rat model, treatment of fucoidan resulted in potent and persistent reduction of high blood pressure (BP) even after drug withdrawal. Our results showed that the mechanisms might involve protection against vascular structure damage, enhanced endothelium-independent vascular function and inhibition of abnormal proliferation of smooth muscle cells, which are mediated by the Akt-eNOS signaling pathway. Moreover, fucoidan treatment reduced the vascular inflammation and oxidative stress control caused by iNOS expression. Together, these results support a putative role of fucoidan in hypertension prevention and treatment. PMID:27153123

  6. RLIP76-dependent suppression of PI3K/AKT/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer

    International Nuclear Information System (INIS)

    MicroRNA-101 (miR-101) participates in carcinogenesis and tumor progression in various cancers. However, its biological functions in prostate cancer are still unclear. Here, we demonstrate that miR-101 represents a critical role in regulating cell apoptosis in prostate cancer cells. We first demonstrated that miR-101 treatment promoted apoptosis in DU145 and PC3 cells by using flow cytometric analysis and transmission electron microscopy (TEM). To verify the mechanisms, we identified a novel miR-101 target, Ral binding protein 1 (RLIP76). We found miR-101 transfection significantly suppresses RLIP76 expression, which can transactivate phosphorylation of PI3K-Akt signaling, and resulted in an amplification of Bcl2-induced apoptosis. Furthermore, we demonstrated that RLIP76 overexpression could reverse the anti-tumor effects of miR-101 in DU145 and PC3 cells by using flow cytometry assay and MTT assay. Taken together, our results revealed that the effect of miR-101 on prostate cancer cell apoptosis was due to RLIP76 regulation of the PI3K/Akt/Bcl-2 signaling pathway. - Highlights: • miR-101 inhibited prostate cancer cell proliferation and enhanced apoptosis. • miR-101 directly targeted and regulated RLIP76 expression. • miR-101 suppressed PI3K/Akt/Bcl-2 signaling pathway by targeting RLIP76

  7. Effect of dehydroepiandrosterone (DHEA) on Akt and protein kinase C zeta (PKCζ) phosphorylation in different tissues of C57BL6, insulin receptor substrate (IRS)1(-/-), and IRS2(-/-) male mice fed a high-fat diet.

    Science.gov (United States)

    Aoki, Kazutaka; Tajima, Kazuki; Taguri, Masataka; Terauchi, Yasuo

    2016-05-01

    We have previously reported that dehydroepiandrosterone (DHEA) suppresses the activity and mRNA expression of the hepatic gluconeogenic enzyme glucose-6-phosphatase (G6Pase), and hepatic glucose production in db/db mice. Tyrosine phosphorylation levels of Insulin receptor substrate (IRS)1 and IRS2 reportedly differ between the liver and muscle tissue and the effect of DHEA on insulin signaling has not been elucidated. Therefore, we examined DHEA's effect on the liver and muscle tissue of IRS1(-/-) and IRS2(-/-) mice. Eight-week-old male C57BL6, IRS1(-/-), and IRS2(-/-) mice were fed a high-fat diet (HFD), or an HFD containing 0.2% DHEA for 4 weeks. In a separate experiment, 8-week-old male C57BL6 mice were fed an HFD or an HFD containing 0.2% androstenedione for 4 weeks. In an insulin tolerance test, DHEA administration decreased the initial plasma glucose levels in the C57BL6, IRS1(-/-), and IRS2(-/-) mice but did not decrease the ratios to the basal blood glucose level. Although DHEA administration increased Akt phosphorylation in the liver of the C57BL6, IRS1(-/-), and IRS2(-/-) mice, androstenedione administration did not increase Akt phosphorylation in the liver of C57BL6 mice. DHEA administration did not increase Akt and PKCζ phosphorylation in the muscle tissue of C57BL6, IRS1(-/-), or IRS2(-/-) mice. However, androstenedione administration increased Akt and PKCζ phosphorylation in the muscle tissue of C57BL6 mice. These findings suggest that the effect of DHEA on insulin action in the liver is self-mediated by DHEA or DHEA sulfate (DHEA-S) in the presence of IRS1, IRS2, or both. PMID:26976654

  8. Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gorgani-Firuzjaee, Sattar [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Adeli, Khosrow [Division of Clinical Biochemistry, The Hospital for Sick Children, University of Toronto, Toronto (Canada); Meshkani, Reza, E-mail: rmeshkani@tums.ac.ir [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of)

    2015-08-21

    The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway.

  9. Osthole suppresses hepatocyte growth factor (HGF)-induced epithelial-mesenchymal transition via repression of the c-Met/Akt/mTOR pathway in human breast cancer cells.

    Science.gov (United States)

    Hung, Chao-Ming; Kuo, Daih-Huang; Chou, Chun-Hung; Su, Yen-Chao; Ho, Chi-Tang; Way, Tzong-Der

    2011-09-14

    Substantial activation of the HGF/c-Met signaling pathway is involved in the progression of several types of cancers and associated with increased tumor invasion and metastatic potential. Underlying HGF-induced tumorigenesis, epithelial to mesenchymal transition (EMT) shows a positive correlation with progression in patients. We previously determined that osthole is a potent fatty acid synthase (FASN) inhibitor. FASN is implicated in cancer progression and may regulate lipid raft function. We therefore examined whether osthole could block HGF-induced tumorigenesis by disrupting lipid rafts. Here, we found that osthole could abrogate HGF-induced cell scattering, migration, and invasion in MCF-7 breast cancer cells. Osthole also effectively inhibited the HGF-induced decrease of E-cadherin and increase of vimentin via down-regulation of phosphorylated Akt and mTOR. Interestingly, osthole blocked HGF-induced c-Met phosphorylation and repressed the expression of total c-Met protein in MCF-7 cells. In addition, C75, a pharmacological inhibitor of FASN, repressed the expression of total c-Met protein in MCF-7 cells. Consistent with a role for FASN, loss of c-Met in cells treated with osthole was prevented by the exogenous addition of palmitate. Briefly, our result suggests a connection between FASN activity and c-Met protein expression and that osthole is a potential compound for breast cancer therapy by targeting the major pathway of HGF/c-Met-induced EMT. PMID:21806057

  10. Lycopene inhibits PDGF-BB-induced retinal pigment epithelial cell migration by suppression of PI3K/Akt and MAPK pathways

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Chi-Ming [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China); Department of Ophthalmology, Cardinal Tien Hospital, Taipei Hsien, Taiwan, ROC (China); Fang, Jia-You [Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan, ROC (China); Lin, Hsin-Huang [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China); Yang, Chi-Yea [Department of Biotechnology, Vanung University, Taoyuan, Taiwan, ROC (China); Hung, Chi-Feng, E-mail: 054317@mail.fju.edu.tw [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China)

    2009-10-09

    Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell-substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation.

  11. Sanguinarine Induces Apoptosis of Human Oral Squamous Cell Carcinoma KB Cells via Inactivation of the PI3K/Akt Signaling Pathway.

    Science.gov (United States)

    Lee, Tae Kyung; Park, Cheol; Jeong, Soon-Jeong; Jeong, Moon-Jin; Kim, Gi-Young; Kim, Wun-Jae; Choi, Yung Hyun

    2016-08-01

    Preclinical Research Sanguinarine, an alkaloid isolated from the root of Sanguinaria canadensis and other plants of the Papaveraceae family, selectively induces apoptotic cell death in a variety of human cancer cells, but its mechanism of action requires further elaboration. The present study investigated the pro-apoptotic effects of sanguinarine in human oral squamous cell carcinoma KB cells. Sanguinarine treatment increased DR5/TRAILR2 (death receptor 5/TRAIL receptor 2) expression and enhanced the activation of caspase-8 and cleavage of its substrate, Bid. Sanguinarine also induced the mitochondrial translocation of pro-apoptotic Bax, mitochondrial dysfunction, cytochrome c release to the cytosol, and activation of caspase-9 and -3. However, a pan-caspase inhibitor, z-VAD-fmk, reversed the growth inhibition and apoptosis induced by sanguinarine. Sanguinarine also suppressed the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt in KB cells, while co-treatment of cells with sanguinarine and a PI3K inhibitor revealed synergistic apoptotic effects. However, pharmacological inhibition of AMP-activated protein kinase and mitogen-activated protein kinases did not reduce or enhance sanguinarine-induced growth inhibition and apoptosis. Collectively, these findings indicate that the pro-apoptotic effects of sanguinarine in KB cells may be regulated by a caspase-dependent cascade via activation of both intrinsic and extrinsic signaling pathways and inactivation of PI3K/Akt signaling. Drug Dev Res 77 : 227-240, 2016.   © 2016 Wiley Periodicals, Inc. PMID:27363951

  12. Tenuigenin Prevents IL-1β-induced Inflammation in Human Osteoarthritis Chondrocytes by Suppressing PI3K/AKT/NF-κB Signaling Pathway.

    Science.gov (United States)

    Wang, Chunlei; Zeng, Lihong; Zhang, Tao; Liu, Jiakun; Wang, Wenbo

    2016-04-01

    Tenuigenin (TEN), the main active component of Polygala tenuifolia, has been reported to have anti-inflammatory effects. However, the effects of TEN on IL-1β-stimulated osteoarthritis chondrocytes have not been reported. The purpose of this study was to investigate the anti-inflammatory effects and mechanism of TEN on IL-1β-stimulated human osteoarthritis chondrocytes. Human osteoarthritis chondrocytes were pretreated with or without TEN for 1 h and then stimulated with IL-1β. The production of NO and PGE2 were detected by the Griess reagent and ELISA. The expression of NF-κB and MAPKs (p38, JNK, ERK) were measured by Western blot analysis. The production of MMP-1, MMP3, and MMP13 were measured by ELISA. The results showed that treatment of TEN significantly inhibited IL-1β-induced NO and PGE2 production. TEN also suppressed IL-1β-induced MMP-1, MMP3, and MMP13 expression. Furthermore, TEN was found to inhibit IL-1β-induced NF-κB activation, PI3K, and AKT phosphorylation. In conclusion, these results suggest that TEN inhibits IL-1β-induced inflammation in human osteoarthritis chondrocytes by inhibiting PI3K/AKT/NF-κB signaling pathway. PMID:26846886

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae, E-mail: chidkim@pusan.ac.kr

    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.

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

    International Nuclear Information System (INIS)

    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. Safflower polysaccharide induces NSCLC cell apoptosis by inhibition of the Akt pathway.

    Science.gov (United States)

    Li, Jian-Ying; Yu, Jun; Du, Xu-Sheng; Zhang, Hui-Min; Wang, Bo; Guo, Hua; Bai, Jie; Wang, Juan-Hong; Liu, An; Wang, Yi-Li

    2016-07-01

    Lung cancer is the leading cause of cancer death in the world. Safflower polysaccharide (SPS) has been used for the improvement of immunomodulatory activities and treatment of cancers. However, studies on the effect of SPS on the progression of lung cancer have rarely been reported. To study the antitumor effect of SPS on human lung cancer and its potential mechanism, non-small cell lung cancer cell lines (NSCLC), A549 and YTMLC-90 were treated with SPS at various concentrations ranging from 0.04 to 2.56 mg/ml and BALB/c nude tumor-bearing mice were injected intraperitoneally with SPS at concentrations ranging from 15 to 135 mg/kg. Results showed that SPS suppressed the proliferation of A549 and YTMLC-90 cells and induced apoptosis by increasing mRNA levels of bax and caspase-3, and inhibited tumor growth in vivo. SPS induced cell cycle arrest in the G2/M phase by decreasing the expression of cdc25B and cyclin B1. Moreover, SPS decreased the expression of Akt, p-Akt and PI3K. In mice, SPS injection enhanced immunomodulatory activities by increasing levels of TNF-α and IL-6 in tumor-bearing mice. Our findings suggest that SPS suppresses tumor growth by enhancing immunomodulatory activities and blocking the PI3K/Akt pathway. This study provides new insight into the anticancer mechanism of SPS. PMID:27177149

  16. TGF-β2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    International Nuclear Information System (INIS)

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-β2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-β2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-β2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-β2 and FGF-2 oppositely affect BCE cell proliferation and TGF-β2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-β2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-β2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-β2-induced suppression of the PI3-kinase/AKT signaling pathway

  17. Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells

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    Hong Sam-Pyo

    2009-02-01

    Full Text Available Abstract Background The Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC. Akt-induced epithelial-to-mesenchymal transition (EMT involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-κB, ERK, and p38. Methods We screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA treatment would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-κB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis. Results Of the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-κB signaling

  18. Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling.

    Directory of Open Access Journals (Sweden)

    Jiaojiao Pang

    Full Text Available The endoplasmic reticulum (ER plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH.ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs. Myocardial mechanical and intracellular Ca(2+ properties, ER stress, autophagy and associated cell signaling molecules were evaluated.ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca(2+ homeostasis, oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62, along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene.Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy.

  19. EGF Prevents the Neuroendocrine Differentiation of LNCaP Cells Induced By Serum Deprivation: The Modulator Role of P13K/Akt

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    Rosa M. Martín-Orozco

    2007-08-01

    Full Text Available The primary focus of this investigation was to study the relationship between neuroendocrine (NE differentiation, epidermal growth factor (EGF because both have been implicated in the progression of prostate cancer. For this purpose, we used gefitinib, trastuzumab, which are inhibitors of EGF receptor (EGFR, ErbB2, respectively. EGF prevents NE differentiation induced by androgen depletion. This effect is prevented by gefitinib, which blocks the activation of EGFR, ErbB2, stimulation of mitogen-activated protein kinase (MAPK, cell proliferation induced by EGF. Conversely, trastuzumab does not inhibit the effect of EGF on EGFR phosphorylation, MAPK activity, cell proliferation, NE differentiation, although it reduces ErbB2 levels specifically, suggesting that ErbB2 is not necessary to inhibit NE differentiation. Prevention of NE differentiation by EGF is mediated by a MAPK-dependent mechanism, requires constitutive Akt activation. The abrogation of the PI3K/Akt pathway changes the role of EGF from inhibitor to inductor of NE differentiation. We show that EGFR tyrosine kinase, MAPK, PI3K inhibitors inhibit the cell proliferation stimulated by EGF but induce the acquisition of NE phenotype. Altogether, the present data should be borne in mind when designing new clinical schedules for the treatment of prostate cancer, including the use of ErbB receptors, associated signaling pathway inhibitors.

  20. Transforming growth factor-β1 regulated phosphorylated AKT and interferon gamma expressions are associated with epithelial cell survival in rhesus macaque colon explants.

    Science.gov (United States)

    Pahar, Bapi; Pan, Diganta; Lala, Wendy; Kenway-Lynch, Carys S; Das, Arpita

    2015-05-01

    Transforming growth factor-β1 (TGF-β1) is an important immunoregulatory cytokine that plays an obligate role in regulating T-cell functions. Here, we demonstrated the role of TGF-β1 in regulating the survival of intestinal epithelial cells (ECs) in rhesus colon explant cultures using either anti-TGF-β1 antibody or recombinant TGF-β1 proteins. Neutralization of endogenous TGF-β1 using anti-TGF-β1 antibodies induced apoptosis of both intestinal ECs and lamina propria (LP) cells. Additionally, endogenous TGF-β1 blocking significantly increased expression of IFNγ, TNFα, CD107a and Perforin in LP cells compared to media and isotype controls. A significant decrease in pAKT expression was detected in anti-TGF-β1 MAbs treated explants compared to isotype and rTGF-β1 protein treated explants. Our results demonstrated TGF-β1 regulated pAKT and IFNγ expressions were associated with epithelial cell survival in rhesus macaque colon explants and suggest a potential role of mucosal TGF-β1 in regulating intestinal homeostasis and EC integrity. PMID:25769244

  1. Downregulation of Akt1 Inhibits Anchorage-Independent Cell Growth and Induces Apoptosis in Cancer Cells

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    Xuesong Liu

    2001-01-01

    Full Text Available The serine/threonine kinases, Akti/PKBα, Akt2/PKBβ, and Akt3/PKBγ, play a critical role in preventing cancer cells from undergoing apoptosis. However, the function of individual Akt isoforms in the tumorigenicity of cancer cells is still not well defined. In the current study, we used an AM antisense oligonucleotide (AS to specifically downregulate Akti protein in both cancer and normal cells. Our data indicate that AM AS treatment inhibits the ability of MiaPaCa-2, H460, HCT-15, and HT1080 cells to grow in soft agar. The treatment also induces apoptosis in these cancer cells as demonstrated by FRCS analysis and a caspase activity assay. Conversely, Akti AS treatment has little effect on the cell growth and survival of normal human cells including normal human fibroblast (NHF, fibroblast from muscle (FBM, and mammary gland epithelial 184135 cells. In addition, AM AS specifically sensitizes cancer cells to typical chemotherapeutic agents. Thus, Akti is indispensable for maintaining the tumorigenicity of cancer cells. Inhibition of AM may provide a powerful sensitization agent for chemotherapy specifically in cancer cells.

  2. Tirofiban induces vasorelaxation of the coronary artery via an endothelium-dependent NO-cGMP signaling by activating the PI3K/Akt/eNOS pathway.

    Science.gov (United States)

    Xia, Tianyang; Guan, Weiwei; Fu, Jinjuan; Zou, Xue; Han, Yu; Chen, Caiyu; Zhou, Lin; Zeng, Chunyu; Wang, Wei Eric

    2016-06-01

    Tirofiban, a glycoprotein IIb/IIIa inhibitor, is an antiplatelet drug extensively used in patients with acute coronary syndrome (ACS) and exerts an therapeutic effect on no-reflow phenomenon during percutaneous coronary intervention (PCI). Previous studies elucidated the vasodilation caused by tirofiban in the peripheral artery. However, whether tirofiban exerts a vasodilator effect on the coronary artery is unclear. Our present study found that tirofiban induced endothelium-dependent vasodilation in a concentration- and time-dependent manner in the isolated rat coronary artery pre-constricted by 5-hydroxytryptamine (5-HT). Further study showed that incubation of human umbilical venous endothelial cells (HUVECs) with tirofiban increased NO production, which was ascribed to the increased eNOS phosphorylation. This was confirmed by the loss of the vasorelaxant effect of tirofiban in the presence of l-NAME (eNOS inhibitor) and L-NMMA (NOS inhibitor) but not SMT (iNOS inhibitor) on isolated rat coronary arteries. The vasorelaxation was also blocked by the PI3K inhibitors, wortmannin and LY294002, as well as the Akt inhibitor SH-5, indicating the role of PI3K and Akt in tirofiban-mediated vasodilation. Moreover, further study showed that soluble guanylyl cyclase (sGC) inhibitor ODQ, or blockers of potassium channel (big-conductance calcium-activated potassium channel) blocked tirofiban-induced vasodilation of the coronary artery. These findings suggest that tirofiban induces vasorelaxation via an endothelium-dependent NO-cGMP signaling through the activation of the Akt/eNOS/sGC pathway. PMID:27018249

  3. Cigarette smoke extract induces COX-2 expression via a PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB pathway and p300 in tracheal smooth muscle cells.

    Science.gov (United States)

    Yang, Chuen-Mao; Lee, I-Ta; Lin, Chih-Chung; Yang, Ya-Lin; Luo, Shue-Fen; Kou, Yu Ru; Hsiao, Li-Der

    2009-11-01

    Exposure to cigarette smoke extract (CSE) leads to airway or lung inflammation, which may be mediated through cyclooxygenase-2 (COX-2) expression and its product prostaglandin E2 (PGE2) synthesis. The aim of this study was to investigate the molecular mechanisms underlying CSE-induced COX-2 expression in human tracheal smooth muscle cells (HTSMCs). Here, we describe that COX-2 induction is dependent on PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB signaling in HTSMCs. CSE stimulated the phosphorylation of c-Src, EGFR, PDGFR, and Akt, which were inhibited by pretreatment with the inhibitor of PKCalpha (Gö6976 or Gö6983), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), or PI3K (LY294002). Moreover, CSE induced a significant increase in COX-2 expression, which was reduced by pretreatment with these inhibitors or transfection with siRNA of PKCalpha, Src, or Akt. Furthermore, CSE-stimulated NF-kappaB p65 phosphorylation and translocation were also attenuated by pretreatment with Gö6976, PP1, AG1478, AG1296, or LY294002. CSE-induced COX-2 expression was also mediated through the recruitment of p300 associated with NF-kappaB in HTSMCs, revealed by coimmunoprecipitation and Western blot analysis. In addition, pretreatment with the inhibitors of NF-kappaB (helenalin) and p300 (garcinol) or transfection with p65 siRNA and p300 siRNA markedly inhibited CSE-regulated COX-2 expression. However, CSE-induced PGE2 generation was reduced by pretreatment with the inhibitor of COX-2 (NS-398). These results demonstrated that in HTSMCs, CSE-induced COX-2-dependent PGE2 generation was mediated through PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt leading to the recruitment of p300 with NF-kappaB complex. PMID:19717552

  4. Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention.

    Directory of Open Access Journals (Sweden)

    Miaozong Wu

    Full Text Available BACKGROUND: Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention. PRINCIPAL FINDINGS: Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308 was higher in soleus muscles of very aged rats (33-months. Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR phosphorylation, along with decreased levels of insulin receptor beta (IR-beta, phosphoinositide 3-kinase (PI3K, phosphatase and tensin homolog deleted on chromosome 10 (PTEN and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1 (Ser241. In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS. Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month animals with acetaminophen (30 mg/kg body weight/day for 6-months. CONCLUSIONS: These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.

  5. Sensitization of Cancer Cells through Reduction of Total Akt and Downregulation of Salinomycin-Induced pAkt, pGSk3β, pTSC2, and p4EBP1 by Cotreatment with MK-2206

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    Ae-Ran Choi

    2014-01-01

    Full Text Available MK-2206 is an inhibitor of Akt activation. It has been investigated as an anticancer drug in clinical trials assessing the potential of pAkt targeting therapy. The purpose of this study was to identify conditions that increase the sensitivity of cancer cells to MK-2206. We found that the treatment of cancer cells with a high concentration of salinomycin (Sal reduced total Akt protein levels but increased activated Akt levels. When cancer cells were cotreated with MK-2206 and Sal, both pAkt and total Akt levels were reduced. Using microscopic observation, an assessment of cleaved PARP, FACS analysis of pre-G1 region, and Hoechst staining, we found that Sal increased apoptosis of MK-2206-treated cancer cells. These results suggest that cotreatment with MK-2206 and Sal sensitizes cancer cells via reduction of both pAkt and total Akt. Furthermore, cotreatment of cancer cells with Sal and MK-2206 reduced pp70S6K, pmTOR, and pPDK1 levels. In addition, Sal-induced activation of GSK3β, TSC2, and 4EBP1 was abolished by MK-2206 cotreatment. These results suggest that cotreatment using MK-2206 and Sal could be used as a therapeutic method to sensitize cancer cells through targeting of the PI3K/Akt/mTOR pathway. Our findings may contribute to the development of MK-2206-based sensitization therapies for cancer patients.

  6. Naringin induces autophagy-mediated growth inhibition by downregulating the PI3K/Akt/mTOR cascade via activation of MAPK pathways in AGS cancer cells.

    Science.gov (United States)

    Raha, Suchismita; Yumnam, Silvia; Hong, Gyeong Eun; Lee, Ho Jeong; Saralamma, Venu Venkatarame Gowda; Park, Hyeon-Soo; Heo, Jeong Doo; Lee, Sang Joon; Kim, Eun Hee; Kim, Jin-A; Kim, Gon Sup

    2015-09-01

    Naringin, one of the major bioflavonoid of Citrus, has been demonstrated as potential anticancer agent. However, the underlying anticancer mechanism still needs to be explored further. This study investigated the inhibitory effect of Naringin on human AGS cancer cells. AGS cell proliferation was inhibited by Naringin in a dose- and time-dependent manner. Naringin did not induce apoptotic cell death, determined by no DNA fragmentation and the reduced Bax/Bcl-xL ratio. Growth inhibitory role of Naringin was observed by western blot analysis demonstrating downregulation of PI3K/Akt/mTOR cascade with an upregulated p21CIPI/WAFI. Formation of cytoplasmic vacuoles and autophagosomes were observed in Naringin-treated AGS cells, further confirmed by the activation of autophagic proteins Beclin 1 and LC3B with a significant phosphorylation of mitogen activated protein kinases (MAPKs). Collectively, our observed results determined that anti-proliferative activity of Naringin in AGS cancer cells is due to suppression of PI3K/Akt/mTOR cascade via induction of autophagy with activated MAPKs. Thus, the present finding suggests that Naringin induced autophagy- mediated growth inhibition shows potential as an alternative therapeutic agent for human gastric carcinoma. PMID:26201693

  7. The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis*

    OpenAIRE

    Oh, Eunjin; Thurmond, Debbie C.

    2006-01-01

    Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [32P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of...

  8. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  9. Protection against 1-methyl-4-phenyl pyridinium-induced neurotoxicity in human neuroblastoma SH-SY5Y cells by Soyasaponin I by the activation of the phosphoinositide 3-kinase/AKT/GSK3β pathway.

    Science.gov (United States)

    Guo, Zheng; Cao, Wei; Zhao, Shifeng; Han, Zengtai; Han, Boxiang

    2016-07-01

    Parkinson's disease (PD) can be ascribed to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta, and thus molecules with neuroprotective ability may have therapeutic value against PD. In the current study, the neuroprotective effects and underlying mechanisms of Soyasaponin I (Soya-I), a naturally occurring triterpene extracted from a widely used ingredient in many foods, such as Glycine max (soybean), were evaluated in a widely used cellular PD model in which neurotoxicity was induced by 1-methyl-4-phenyl pyridinium (MPP) in cultured SH-SY5Y cells. We found that Soya-I at 10-40 μM considerably protected against MPP-induced neurotoxicity as evidenced by an increase in cell viability, a decrease in lactate dehydrogenase release, and a reduction in apoptotic nuclei. Moreover, Soya-I effectively inhibited the elevated intracellular accumulation of reactive oxygen species as well as the Bax/Bcl-2 ratio caused by MPP. Most importantly, Soya-I markedly reversed the inhibition of protein expression of phosphorylated AKT and phosphorylated GSK3β caused by MPP. LY294002, the specific inhibitor of phosphoinositide 3-kinase, significantly abrogated the upregulated phosphorylated AKT and phosphorylated GSK3β offered by Soya-I, suggesting that the neuroprotection of Soya-I was mainly dependent on the activation of the phosphoinositide 3-kinase/AKT/GSK3β signaling pathway. The results taken together indicate that Soya-I may be a potential candidate for further preclinical study aimed at the prevention and treatment of PD. PMID:27196724

  10. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    International Nuclear Information System (INIS)

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility

  11. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Jianwei [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China)

    2015-08-14

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.

  12. Cisplatin Induces Overactivation of the Dormant Primordial Follicle through PTEN/AKT/FOXO3a Pathway which Leads to Loss of Ovarian Reserve in Mice.

    Directory of Open Access Journals (Sweden)

    Eun Mi Chang

    Full Text Available Cisplatin is a first-line chemotherapeutic agent for ovarian cancer that acts by promoting DNA cross links and adduct. However drug resistance and considerable side effects including reproductive toxicity remain a significant challenge. PTEN is well known as a tumor suppressor function which plays a fundamental role in the regulation of the cell cycle, apoptosis and development of cancer. At the same time PTEN has been revealed to be critically important for the maintenance of the primordial follicle pool. In this study, we investigated the role of PTEN/Akt/FOXO3 pathway in cisplatin-induced primordial follicle depletion. Cisplatin induced ovarian failure mouse model was used to evaluate how this pathway involves. In vitro maturation was used for oocyte rescue after cisplatin damage. We found that cisplatin treatment decreased PTEN levels, leading to a subsequent increase in the phosphorylation of key molecules in the pathway. The activation of the PTEN/Akt/FOXO3 pathway cascade increased cytoplasmic translocation of FOXO3a in cisplatin-treated follicles, which in turn increased the pool size of growing follicles, and rapidly depleted the number of dormant follicles. Once activated, the follicles were more prone to apoptosis, and their cumulus cells showed a loss of luteinizing hormone (LH receptor expression, which leads to failure during final maturation and ovulation. In vitro maturation to rescue oocytes in a cisplatin-treated mouse model resulted in successful maturation and fertilization. This study is the first to show the involvement of the PTEN/Akt/FOXO3 pathway in premature ovarian failure after cisplatin treatment and the possibility of rescue through in vitro maturation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio, E-mail: yyoneda@p.kanazawa-u.ac.jp

    2014-07-18

    Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

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

    International Nuclear Information System (INIS)

    Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5Rgsc451 mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway

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

    International Nuclear Information System (INIS)

    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

  16. Phorbol ester and light-induced endogenous phosphorylation of rat retinal rod outer segment (ROS) proteins

    International Nuclear Information System (INIS)

    The authors have previously described the presence of a C-kinase in bovine ROS and its in vitro activation by light in crude rat ROS. In this study, they have labelled the retinas with 33Pi by intravitreal injection and compared the phosphorylation pattern of ROS proteins induced by light and activators of the C-kinase phosphorylation system. Except for light treatment, all procedures were carried out in complete darkness using an infrared image converter. Incubation of 33P-labelled retinas in light for 5 minutes resulted in the phosphorylation of rhodopsin, 80,65,47,44, and 15kd proteins of crude ROS. Incubation of 33P-labelled retinas with 5μM 12-0-tetradecanoylphorbol-13-acetate (TPA) resulted in the phosphorylation of 80,65,47,44,33, and 15 kd proteins of crude ROS. The complete darkness control did not exhibit any phosphorylation or proteins whereas the red light control exhibited variable low phosphorylation of 80,47,44, and 15kd proteins. 1-oleoyl-2-acetyl-glycerol (OAG) at 500μg/ml caused the phosphorylation of the same proteins as observed with TPA. TPA (0.5-500μM) and OAG (150-500μg/ml) did not induce rhodopsin phosphorylation. Since light, TPA and OAG exhibit similarities in the phosphorylation patterns of proteins (except for rhodopsin), these results suggest at least a partial linkage of light and C-kinase effects in vivo

  17. PAR1- and PAR2-induced innate immune markers are negatively regulated by PI3K/Akt signaling pathway in oral keratinocytes

    Directory of Open Access Journals (Sweden)

    Dale Beverly A

    2010-10-01

    Full Text Available Abstract Background Protease-Activated Receptors (PARs, members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs, but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. Results Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. Conclusion Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.

  18. HDAC inhibition elicits myocardial protective effect through modulation of MKK3/Akt-1.

    Directory of Open Access Journals (Sweden)

    Ting C Zhao

    Full Text Available We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3⁻/⁻, Akt-1⁻/⁻ mice received intraperitoneal injections of trichostatin A (0.1 mg/kg, a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection.

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

    Science.gov (United States)

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

    2016-05-01

    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 months) and aged (20 months) Sprague Dawley (SD) rats were subjected to MI/Rin 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

  20. Quercetin 3-O-methyl ether protects FL83B cells from copper induced oxidative stress through the PI3K/Akt and MAPK/Erk pathway

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Hsiao-Ling, E-mail: lily1001224@gmail.com [Department of Life Sciences, Tzu Chi University, Hualien, Taiwan (China); Li, Chia-Jung, E-mail: 97751101@stmail.tcu.edu.tw [Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan (China); Huang, Lin-Huang, E-mail: yg1236@yahoo.com.tw [School of Medicine, Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan (China); Chen, Chun-Yao, E-mail: cychen@mail.tcu.edu.tw [Department of Life Sciences, Tzu Chi University, Hualien, Taiwan (China); Tsai, Chun-Hao, E-mail: 100726105@stmail.tcu.edu.tw [Department of Life Sciences, Tzu Chi University, Hualien, Taiwan (China); Lin, Chun-Nan, E-mail: lincna@cc.kmu.edu.tw [Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Department of Biological Science and Technology, School of Medicine, China Medical University, Taichung, Taiwan (China); Hsu, Hsue-Yin, E-mail: hsueyin@mail.tcu.edu.tw [Department of Life Sciences, Tzu Chi University, Hualien, Taiwan (China)

    2012-10-01

    Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu{sup 2+}-induced cytotoxicity. Exposure to Cu{sup 2+} resulted in the death of mouse liver FL83B cells, characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu{sup 2+}-induced apoptosis and mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu{sup 2+}-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (MnSOD) was shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu{sup 2+} at a concentration of 5 μM. Hepatic damage induced by Cu{sup 2+} was reduced by cotreatment with Q3. Survival of Cu{sup 2+}-exposed larval zebrafish was significantly increased by cotreatment with 15 μM Q3. Our results indicated that Cu{sup 2+}-induced apoptosis in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, overexpression of 14-3-3, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes. Highlights: ► Protective effects of Q3 on Cu{sup 2+}-induced oxidative stress in vitro and in vivo. ► Cu{sup 2+} induced apoptosis in FL83B cells via ROS and the activation of Erk. ► Q3 abolishes Cu{sup 2+}-induced apoptosis through the PI3K/Akt and MAPK

  1. Propofol pretreatment attenuates lipopolysaccharide-induced acute lung injury in rats by activating the phosphoinositide-3-kinase/Akt pathway

    International Nuclear Information System (INIS)

    The aim of this study was to investigate the effect of propofol pretreatment on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the role of the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway in this procedure. Survival was determined 48 h after LPS injection. At 1 h after LPS challenge, the lung wet- to dry-weight ratio was examined, and concentrations of protein, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) were determined using the bicinchoninic acid method or ELISA. Lung injury was assayed via lung histological examination. PI3K and p-Akt expression levels in the lung tissue were determined by Western blotting. Propofol pretreatment prolonged survival, decreased the concentrations of protein, TNF-α, and IL-6 in BALF, attenuated ALI, and increased PI3K and p-Akt expression in the lung tissue of LPS-challenged rats, whereas treatment with wortmannin, a PI3K/Akt pathway specific inhibitor, blunted this effect. Our study indicates that propofol pretreatment attenuated LPS-induced ALI, partly by activation of the PI3K/Akt pathway

  2. Propofol pretreatment attenuates lipopolysaccharide-induced acute lung injury in rats by activating the phosphoinositide-3-kinase/Akt pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, L.L. [Department of Anesthesiology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province (China); Hu, G.C. [Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, IL (United States); Zhu, S.S. [Department of Anesthesiology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province (China); Li, J.F. [Department of Anesthesiology, Tengzhou Central People' s Hospital, Liaocheng, Shandong Province (China); Liu, G.J. [Department of Anesthesiology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province (China)

    2014-10-14

    The aim of this study was to investigate the effect of propofol pretreatment on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the role of the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway in this procedure. Survival was determined 48 h after LPS injection. At 1 h after LPS challenge, the lung wet- to dry-weight ratio was examined, and concentrations of protein, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) were determined using the bicinchoninic acid method or ELISA. Lung injury was assayed via lung histological examination. PI3K and p-Akt expression levels in the lung tissue were determined by Western blotting. Propofol pretreatment prolonged survival, decreased the concentrations of protein, TNF-α, and IL-6 in BALF, attenuated ALI, and increased PI3K and p-Akt expression in the lung tissue of LPS-challenged rats, whereas treatment with wortmannin, a PI3K/Akt pathway specific inhibitor, blunted this effect. Our study indicates that propofol pretreatment attenuated LPS-induced ALI, partly by activation of the PI3K/Akt pathway.

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

    International Nuclear Information System (INIS)

    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 α 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.

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

    Science.gov (United States)

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

    2012-05-01

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

  5. Deletion of the N-terminus of IKKγ induces apoptosis in keratinocytes and impairs the AKT/PTEN signaling pathway

    International Nuclear Information System (INIS)

    The regulatory subunit IKKγ/NEMO is crucial for skin development and function and although devoid of kinase activity, loss of IKKγ function completely abolishes the activation of NF-κB by all pro-inflammatory cytokines. To inhibit the IκB kinase (IKK) complex in keratinocytes, we have used a dominant negative approach by generating stable transfectants of an N-terminal deletion of IKKγ (IKKγ-DN97) that uncouples formation of the IKK complex. Expression of this mutant in PB keratinocytes (PB-IKKγ-DN97) delayed growth kinetics, caused morphological changes and dramatically augmented apoptosis even in the absence of pro-apoptotic stimuli, as determined by cell morphology, TUNEL and caspase-3 cleavage. Moreover, in PB-IKKγ-DN97 cells, TNF-α and IL-1 treatment failed to induce degradation of IκBα, phosphorylation of p65 on Ser 536 and nuclear translocation which, consequently, reduced κB-binding activity. In PB-IKKγ-DN97 cells, accumulation of IκBα correlated with a downregulation of AKT activity and an increase of PTEN protein levels whereas pro-apoptotic p53 target genes Bax and Puma were upregulated. These effects were most likely mediated through IKK since coexpression of the wild-type form of IKKγ in keratinocytes partially reversed apoptosis and reduced PTEN expression. Thus, our data suggest a negative cross-talk mechanism involving PTEN and NF-κB, critical for the anti-apoptotic role of NF-κB in keratinocytes

  6. 骨骼肌收缩模式对p38/Akt磷酸化水平的影响%The influence of contraction modes on the phosphorylation of p38/Akt

    Institute of Scientific and Technical Information of China (English)

    李辉; 焦博; 余志斌; 陈自谦

    2011-01-01

    Objective: Muscle contraction may prompt glucose uptake through non-insulin-dependent ways, and it may be due to the enhanced activation of key proteins known to regulate glucose metabolism, like p38 and Akt. Our experiment focused on the impact of different contraction modes on the phosphorylation of the molecules, thus to explore effective ways to lower blood glucose. Methods: Isolated muscle strips perfusion technique and Western blot analysis were employed to investigate the influence of different modes of contraction on the activation of the molecules. Results: Muscle contraction led to an increase in p38 phosphorylation, with the greatest effect observed after 5 minutes of 10% DC(duty cycle) contraction and 5 minutes of 1 % DC contraction. However, phosphorylation of Akt were not altered by the two contraction modes. Conclusion: The level of phosphorylation of p38 was higher at the optimal contraction modes, but these modes could not increase the level of phosphorlation of Akt.%目的:骨骼肌收缩可能通过非胰岛素依赖的途径促进葡萄糖摄取,而p38与Akt可能是其中起重要作用的分子.本文研究骨骼肌不同收缩模式对上述信号分子磷酸化的影响,从而探讨有效降低血糖的运动方式.方法:采用离体比目鱼肌肌条灌流技术及Western blot检测方法,研究不同模式的收缩对骨骼肌p38、Akt磷酸化水平的影响.结果:5 min 10%DC(duty cycle负荷率)和5min 1% DC的收缩模式可分别使p38的磷酸化较对照组增加30%和34%,是激活p38的适宜刺激.但对Akt的磷酸化水平没有影响.结论:低强度有氧运动可以更好地激活p38,但不能有效激活Akt.

  7. Anger Emotional Stress Influences VEGF/VEGFR2 and Its Induced PI3K/AKT/mTOR Signaling Pathway.

    Science.gov (United States)

    Sun, Peng; Wei, Sheng; Wei, Xia; Wang, Jieqiong; Zhang, Yuanyuan; Qiao, Mingqi; Wu, Jibiao

    2016-01-01

    Objective. We discuss the influence of anger emotional stress upon VEGF/VEGFR2 and its induced PI3K/AKT/mTOR signal pathway. Methods. We created a rat model of induced anger (anger-out and anger-in) emotional response using social isolation and resident-intruder paradigms and assessed changes in hippocampus' VEGF content, neuroplasticity, and the PI3K/AKT/mTOR signaling pathway. Results. The resident-intruder method successfully generated anger-out and anger-in models that differed significantly in composite aggression score, aggression incubation, open field behavior, sucrose preference, and weight gain. Anger emotional stress decreased synaptic connections and VEGFR2 expression. Anger emotional stress led to abnormal expression of VEGF/VEGFR2 mRNA and protein and disorderly expression of key factors in the PI3K/AKT/mTOR signal pathway. Fluoxetine administration ameliorated behavioral abnormalities and damage to hippocampal neurons caused by anger emotional stress, as well as abnormal expression of some proteins in VEGF/VEGFR2 and its induced PI3K/AKT/mTOR signal pathway. Conclusion. This research provides a detailed classification of anger emotion and verifies its influence upon VEGF and the VEGF-induced signaling pathway, thus providing circumstantial evidence of mechanisms by which anger emotion damages neurogenesis. As VEGFR2 can promote neurogenesis and vasculogenesis in the hippocampus and frontal lobe, these results suggest that anger emotional stress can result in decreased neurogenesis. PMID:27057362

  8. Upregulation of p‑Akt by glial cell line‑derived neurotrophic factor ameliorates cell apoptosis in the hippocampus of rats with streptozotocin‑induced diabetic encephalopathy.

    Science.gov (United States)

    Cui, Weigang; Zhang, Yinghua; Lu, Derong; Ren, Mingxin; Yuan, Guoyan

    2016-01-01

    The loss of neurotrophic factor support has been shown to contribute to the development of the central nervous system. Glial cell line‑derived neurotrophic factor (GDNF), a potent neurotrophic factor, is closely associated with apoptosis and exerts neuroprotective effects on numerous populations of cells. However, the underlying mechanisms of these protective effects remain unknown. In the present study, a significant increase in Bax levels and DNA fragmentation was observed in the hippocampus obtained from the brains of diabetic rats 60 days after diabetes had been induced. The apoptotic changes were correlated with the loss of GDNF/Akt signaling. GDNF administration was found to reverse the diabetes‑induced Bax and DNA fragmentation changes. This was associated with an improvement in the level of p‑Akt/Akt. In addition, combination of GDNF with a specific inhibitor of the phosphoinositide 3‑kinase (PI3K)/Akt pathway, Wortmannin, significantly abrogated the effects of GDNF on the levels of p‑Akt/Akt, Bax and DNA fragmentation. However, a p38 mitogen‑activated proten kinase (MAPK) inhibitor, SB203580, had no effect on the expression of p‑Akt/Akt, Bax or DNA fragmentation. These results demonstrate the pivotal role of GDNF as well as the PI3K/Akt pathway, but not the MAPK pathway, in the prevention of diabetes‑induced neuronal apoptosis in the hippocampus. PMID:26549420

  9. Novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivative, MHY-449, induces cell cycle arrest and apoptosis via the downregulation of Akt in human lung cancer cells.

    Science.gov (United States)

    Lim, Hyun Sook; Kang, Yong Jung; Sung, Bokyung; Kim, Seon Hee; Kim, Min Jeong; Kim, Hye Rim; Kim, Seong Jin; Choi, Yung Hyun; Moon, Hyung Ryong; Chung, Hae Young; Kim, Nam Deuk

    2015-11-01

    The anticancer properties of MHY-449, a novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivative, in various human cancer cell lines have been previously reported. The aim of the present study was to investigate the activities of MHY-449 on human lung cancer cells in order to elucidate its underlying molecular mechanisms of action. The result showed that MHY-449 treatment inhibited cell growth in a time- and concentration‑dependent manner. Specifically, MHY-449 induced cell cycle arrest at the S phase, and the resulting increased sub-G1 fraction led to the induction of apoptosis, as determined by flow cytometric analysis and DNA fragmentation. In addition, MHY-449 was shown to induce alterations in the ratio of Bax/Bcl-2 protein expression, and contribute to the loss of mitochondrial membrane potential. These cellular events then triggered the caspase cascade and subsequent poly(ADP‑ribose) polymerase cleavage. The apoptotic cell death induced by MHY-449 was inhibited by pretreatment with Z-VAD‑FMK, a pan-caspase inhibitor. Moreover, MHY-449 downregulated the phosphorylation of Akt, and the phosphatidylinositol-3 kinase/Akt inhibitor LY294002 was found to enhance its induction of apoptosis. Taken together, the results suggested that MHY-449 exerts anticancer effects by promoting cell cycle arrest and apoptosis via the downregulation of Akt. Based on these data, MHY-449 serves as a potential candidate in the chemoprevention and/or treatment of lung cancer. PMID:26324335

  10. Akt Switches TopBP1 Function from Checkpoint Activation to Transcriptional Regulation through Phosphoserine Binding-Mediated Oligomerization

    OpenAIRE

    Liu, Kang; Graves, Joshua D.; Scott, Jessica D.; Li, Rongbao; Lin, Weei-Chin

    2013-01-01

    Our previous study showed that Akt phosphorylates TopBP1 at the Ser-1159 residue and induces its oligomerization. Oligomerization is required for TopBP1 to bind and repress E2F1 activity. However, the mechanism through which phosphorylation of TopBP1 by Akt leads to its oligomerization remains to be determined. Here, we demonstrate that binding between the phosphorylated Ser-1159 (pS1159) residue and the 7th and 8th BRCT domains of TopBP1 mediates TopBP1 oligomerization. Mutations within the ...

  11. Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.

    Science.gov (United States)

    Matusiak, Magdalena; Van Opdenbosch, Nina; Vande Walle, Lieselotte; Sirard, Jean-Claude; Kanneganti, Thirumala-Devi; Lamkanfi, Mohamed

    2015-02-01

    The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5. PMID:25605939

  12. Lutein Inhibits the Migration of Retinal Pigment Epithelial Cells via Cytosolic and Mitochondrial Akt Pathways (Lutein Inhibits RPE Cells Migration

    Directory of Open Access Journals (Sweden)

    Ching-Chieh Su

    2014-08-01

    Full Text Available During the course of proliferative vitreoretinopathy (PVR, the retinal pigment epithelium (RPE cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2011-02-01

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

  15. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Lan; Josifi, Erlena; Tiao, Joshua R. [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  16. Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

    OpenAIRE

    Baik, Jiseok; Ok, Seong-Ho; Cho, Hyunhoo; Yu, Jongsun; Kim, Woochan; Nam, In-Koo; Choi, Mun-Jeoung; Lee, Heon-Keun; Sohn, Ju-Tae

    2014-01-01

    Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in ...

  17. Alzheimer—like phosphorylation of tau and neurofilament induced by cocaine in vivo

    Institute of Scientific and Technical Information of China (English)

    LIUShi-Jie; FANGZheng-Yu; YANGYing; DENGHeng-Mei; WANGJian-Zhi

    2003-01-01

    AIM:To explore the relationship between cocaine-induced cyclin-dependent kinase-5(CDK5) overexpression or overactivation and Alzheimer-like hyperphosphorylation of cytoskeletal protein. METHODS: Cocaine was injected (ip,20mg·kg-1·d-1) into rats and the phosphorylation of neuronal cytoskeletal proteins was measured by Western blotting.RESULTS:The levels of phosphorylated tau at PHF-1 epitope and phosphorylated neurofilament determined by SMI31 were elevated in rat brain hippocampus, cortex, and caudatoputamen on d 8 and d 16 after the injection of cocaine, when compared with saline control rat at the same brain regions. On the other hand, the levels of tau non-phosphorylated at tau-1 site and non-phosphorylated neurofilament determined by SIM32 were decreased in same brain regions at the same time points examined. No significant difference of phosphorylated tau and neurofilament at those epitopes was seen on d 4. Although cocaine injection could induce significant hyperphosphorylation of neuronal cytoskeletal proteins, the overexpression of CDK5 and p35 was not detected. CONCLUSION:Peritoneal injection of cocaine induces Alzheimer-like hyperphosphorylation of tau and neurofilament in rat brain, and the effect may be not relevant to an increase in overexpression or overactivation of CDK5.

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

    Energy Technology Data Exchange (ETDEWEB)

    Oommen, Deepu, E-mail: oommen1978@gmail.com [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland (United Kingdom); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland (United Kingdom)

    2012-05-11

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

  19. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

    International Nuclear Information System (INIS)

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

  20. Neuroinflammation is not a prerequisite for diabetes-induced tau phosphorylation

    Directory of Open Access Journals (Sweden)

    Judith M Van Der Harg

    2015-11-01

    Full Text Available Abnormal phosphorylation and aggregation of tau is a key hallmark of Alzheimer's disease (AD. AD is a multifactorial neurodegenerative disorder for which Diabetes Mellitus (DM is a risk factor. In animal models for DM, the phosphorylation and aggregation of tau is induced or exacerbated, however the underlying mechanism is unknown. In addition to the metabolic dysfunction, DM is characterized by chronic low-grade inflammation. This was reported to be associated with a neuroinflammatory response in the hypothalamus of DM animal models. Neuroinflammation is also implicated in the development and progression of AD. It is unknown whether DM also induces neuroinflammation in brain areas affected in AD, the cortex and hippocampus. Here we investigated whether neuroinflammation could be the mechanistic trigger to induce tau phosphorylation in the brain of DM animals. Two distinct diabetic animal models were used; rats on free-choice high-fat high-sugar (fcHFHS diet that are insulin resistant and streptozotocin-treated rats that are insulin deficient. The streptozotocin-treated animals demonstrated increased tau phosphorylation in the brain as expected, whereas the fcHFHS diet fed animals did not. Remarkably, neither of the diabetic animal models showed reactive microglia or increased GFAP and COX-2 levels in the cortex or hippocampus. From this, we conclude: 1. DM does not induce neuroinflammation in brain regions affected in AD, and 2. Neuroinflammation is not a prerequisite for tau phosphorylation. Neuroinflammation is therefore not the mechanism that explains the close connection between DM and AD.

  1. Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Skov, S;

    1994-01-01

    stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL-2 ligation, whereas...... an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL-2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that...... IL-2 induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84-kDa protein in antigen-specific human T cell lines....

  2. Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.

    Directory of Open Access Journals (Sweden)

    Shu Xiao

    Full Text Available Nucleolin (NCL is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2 and mitotic cyclin-dependent kinase 1 (CDK1. Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2 homology 3 (BH3-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

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

    Science.gov (United States)

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

    2011-11-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  5. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance

    Directory of Open Access Journals (Sweden)

    Euan A. Stronach

    2011-11-01

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

  6. A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration.

    Directory of Open Access Journals (Sweden)

    James D Glover

    Full Text Available In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.

  7. The Akt switch model: Is location sufficient?

    Science.gov (United States)

    Gray, Catheryn W; Coster, Adelle C F

    2016-06-01

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

  8. Anthocyanins Downregulate Lipopolysaccharide-Induced Inflammatory Responses in BV2 Microglial Cells by Suppressing the NF-κB and Akt/MAPKs Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Yung Hyun Choi

    2013-01-01

    Full Text Available Anthocyanins are naturally occurring polyphenols that impart bright color to fruits, vegetables and plants and have a variety of protective properties, which have generally been attributed to their antioxidant capacity. However, little is known about the molecular mechanisms underlying anti-inflammatory effects of anthocyanins related to neurodegenerative diseases. Therefore, we determined whether anthocyanins isolated from black soybean seed coats would inhibit pro-inflammatory mediators and cytokines in lipopolysaccharide (LPS-stimulated murine BV2 microglial cells. Our results showed that anthocyanins significantly inhibited LPS-induced pro-inflammatory mediators, such as nitric oxide (NO and prostaglandin E2, and pro-inflammatory cytokines including tumor necrosis factor (TNF-α and interleukin (IL-1β, without significant cytotoxicity. Anthocyanins also downregulated excessive expression of inducible NO synthase, cyclooxygenase-2, TNF-α, and IL-1β in LPS-stimulated BV2 cells. Moreover, anthocyanins inhibited nuclear translocation of nuclear factor-kappa B (NF-κB by reducing inhibitor of NF-κB alpha degradation as well as phosphorylating extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and Akt. These findings suggest that anthocyanins may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation.

  9. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    Science.gov (United States)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  10. Curcumin induces autophagy, inhibits proliferation and invasion by downregulating AKT/mTOR signaling pathway in human melanoma cells.

    Science.gov (United States)

    Zhao, Guangming; Han, Xiaodong; Zheng, Siwen; Li, Zhen; Sha, Yang; Ni, Jing; Sun, Zhe; Qiao, Song; Song, Zhiqi

    2016-02-01

    Melanoma is the foremost malignant cutaneous cancer and it is extremely resistant to chemotherapy and radiotherapy. Curcumin is an active component of turmeric, the yellow spice derived from the rhizome of Curcuma longa, and is widely known for its anti-inflammatory and anti-cancerogenic properties. Several recent studies suggest that curcumin induces apoptosis by modulating multiple signaling pathways to exert its anticancer effect. In the present study, we investigated the effect of curcumin on the viability, invasion potential, cell cycle, autophagy and the AKT, mTOR, P70S6K proteins of AKT/mTOR signaling pathway in human melanoma A375 and C8161 cell lines in vitro and in an in vivo tumorigenesis model. Curcumin effectively inhibited the proliferation of melanoma cells in vitro and in vivo. It suppressed cell invasion, arrested the cancer cells at G2/M phase of the cell cycle, and induced autophagy. Furthermore, curcumin suppressed the activation of AKT, mTOR and P70S6K proteins. Curcumin, therefore, is a potent suppressor of cell viability and invasion, and simultaneously an inducer of autophagy in A375 and C8161 cells. Accordingly, curcumin could be a novel therapeutic candidate for the management of melanoma. PMID:26573768

  11. Trimetazidine Protects Umbilical Cord Mesenchymal Stem Cells Against Hypoxia and Serum Deprivation Induced Apoptosis by Activation of Akt

    Directory of Open Access Journals (Sweden)

    Xuhe Gong

    2014-12-01

    Full Text Available Background: Mesenchymal stem cell (MSC transplantation is a promising therapy for cardiac repair. However, the efficacy is limited by the poor viability of MSCs in the infarcted heart. Recent findings have implicated that trimetazidine (TMZ enhanced the survival of the stem cells under various conditions. However, as the stem cells in these studies were animal-derived, little information is available about the effects of TMZ on human MSCs. Herein, we propose that TMZ may protect human MSCs against apoptosis induced by Hypoxia/Serum deprivation (H/SD. Methods: Human umbilical cord MSCs (UC-MSCs from Wharton's jelly were pretreated with 10µM TMZ of H/SD with or without the Akt inhibitor LY294002. The morphological changes were assessed using Hoechst 33342. Apoptosis was evaluated via Annexin V/PI staining; and apoptosis-related proteins were detected using Western-blot. Protein chip technology was used to screen for differences between the cell supernatants. Results: TMZ had a significant protective effect against H/SD-induced apoptosis, accompanied by an increase in Bcl-2 and p-Akt. The TMZ-mediated anti-apoptotic effect on MSCs could be attenuated by treatment with LY294002. Moreover, protein chip assays showed that TMZ treatment increased the paracrine functions of MSCs. Conclusion: Trimetazidine protects human UC-MSCs from H/SD-induced apoptosis via the Akt pathway and may therefore be a potentially useful therapeutic adjunct for transplanting MSCs into damaged heart after myocardial infarction.

  12. Structural basis of how stress-induced MDMX phosphorylation activates p53.

    Science.gov (United States)

    Chen, X; Gohain, N; Zhan, C; Lu, W-Y; Pazgier, M; Lu, W

    2016-04-14

    The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage. PMID:26148237

  13. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [32P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  14. Phosphorylation dynamics of radixin in hypoxia-induced hepatocyte injury.

    Science.gov (United States)

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

    2015-02-15

    The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury. PMID:25501552

  15. sPLA2-IIA Augments Oxidized LDL-Induced MCP-1 Expression in Vitro Through Activation of Akt

    Directory of Open Access Journals (Sweden)

    Yongjun Guo

    2015-10-01

    Full Text Available Background/Aims: Group IIA secretory phospholipase A2 (sPLA2-IIA has an important role in atherosclerosis. In this study, we explored whether sPLA2-IIA overexpression could promote atherosclerosis in normal environment alone or with other inflammatory factors. Methods: Human aortic smooth muscle cells (HASMCs were transduced with Lv-GFP-sPLA2-IIA, a plasmid containing sPLA2-IIA coupled with green fluorescent protein (GFP. Cells were incubated in the presence or absence of oxidized low-density lipoprotein (LDL, sPLA2 inhibitor LY315920 or PI3K/Akt inhibitor LY294002. The mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1 were assessed by quantitative real-time polymerase chain reaction (QRT-PCR and enzyme-linked immunosorbent assay (ELISA, respectively. Phosphorylation of Akt was examined by western blotting. Results: Lv-GFP-sPLA2-IIA-transduced HASMCs remained fluorescent during 72 h of the study period with infection ratio of around 80%. The mRNA expression and protein secretion of MCP-1was not altered in groups of HASMCs, Lv-GFP transduced and Lv-GFP-sPLA2-IIA-transduced HASMCs (p>0.05, but was significantly increased in the presence of oxidized LDL especially in Lv-GFP-sPLA2-IIA transduction group (ppConclusions: Overexpression of sPLA2-IIA does not alter MCP-1 level at baseline, but could enhance the atherogenic effect of oxidized LDL in HASMCs, at least partly due to activation of Akt. These findings may provide a strategy for treatment of inflammatory cardiovascular diseases.

  16. Optimal Classes of Chemotherapeutic Agents Sensitized by Specific Small-Molecule Inhibitors of Akt In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Yan Shi

    2005-11-01

    Full Text Available Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654. Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.

  17. Tyrosine 402 phosphorylation of Pyk2 is involved in ionomycin-induced neurotransmitter release.

    Directory of Open Access Journals (Sweden)

    Zhao Zhang

    Full Text Available Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2, in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402 in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F. In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.

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

    Energy Technology Data Exchange (ETDEWEB)

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I. [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Figueroa, Carlos D. [Department of Anatomy, Histology and Pathology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); González, Carlos B., E-mail: cbgonzal@uach.cl [Department of Physiology, Universidad Austral de Chile, Valdivia 509-9200 (Chile); Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2013-11-29

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

  19. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    International Nuclear Information System (INIS)

    Research highlights: → We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. → Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. → This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  20. Protein kinase C {alpha} activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

    DEFF Research Database (Denmark)

    Thomassen, Martin; Rose, Adam John; Jensen, Thomas Elbenhardt;

    2011-01-01

    Exercise induced phosphorylation of FXYD1 is a potential important regulator of Na(+), K(+) pump activity. It was investigated if skeletal muscle contractions induce phosphorylation of FXYD1 and if Protein Kinase C a (PKCa) activity is a prerequisite for this possible mechanism. In part 1, human ...

  1. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    International Nuclear Information System (INIS)

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  2. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  3. Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERK and JNK signaling pathways in human lung cancer cell lines.

    Science.gov (United States)

    Hsieh, Ming-Ju; Tsai, Te-Lung; Hsieh, Yih-Shou; Wang, Chau-Jong; Chiou, Hui-Ling

    2013-11-01

    Our previous study has revealed that dioscin, a compound with anti-inflammatory, lipid-lowering, anticancer and hepatoprotective effects, may induce autophagy in hepatoma cells. Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. In this study, the role of autophagy and related signaling pathways during dioscin-induced apoptosis in human lung cancer cells was investigated. Results from 4'-6-diamidino-2-phenylindole and annexin-V/PI double-staining assay showed that caspase-3- and caspase-8-dependent, and dose-dependent apoptoses were detected after a 24-h dioscin treatment. Meanwhile, autophagy was detected as early as 12 h after an exposure to low-dose dioscin, as indicated by an up-regulated expression of LC3-II and beclin-1 proteins. Blockade of autophagy with bafilomycin A1 or 3-methyladenine sensitized the A549 and H1299 cells to apoptosis. Treatment of A549 and H1299 cells with dioscin caused a dose-dependent increase in ERK1/2 and JNK1/2 activity, accompanied with a decreased PI3K expression and decreased phosphorylation of Akt and mTOR. Taken together, this study demonstrated for the first time that autophagy occurred earlier than apoptosis during dioscin-induced human lung cancer cell line apoptosis. Dioscin-induced autophagy via ERK1/2 and JNK1/2 pathways may provide a protective mechanism for cell survival against dioscin-induced apoptosis to act as a cytoprotective reaction. PMID:23552851

  4. Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma

    Science.gov (United States)

    Villar, Victor Hugo; Vögler, Oliver; Barceló, Francisca; Martín-Broto, Javier; Martínez-Serra, Jordi; Ruiz-Gutiérrez, Valentina; Alemany, Regina

    2016-01-01

    Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5–50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6–9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10–15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS. PMID:27219337

  5. Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma.

    Directory of Open Access Journals (Sweden)

    Victor Hugo Villar

    Full Text Available Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA, being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR, in human soft tissue sarcoma cells. UA (5-50 μM strongly inhibited (up to 80% the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6-9 h strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10-15 μM enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS.

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

    Science.gov (United States)

    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

  7. Marine lipopeptide Iturin A inhibits Akt mediated GSK3β and FoxO3a signaling and triggers apoptosis in breast cancer.

    Science.gov (United States)

    Dey, Goutam; Bharti, Rashmi; Dhanarajan, Gunaseelan; Das, Subhasis; Dey, Kaushik Kumar; Kumar, B N Prashanth; Sen, Ramkrishna; Mandal, Mahitosh

    2015-01-01

    Akt kinase is a critical component of the PI3K/Akt signaling pathway, which is frequently over expressed in human cancers including breast. Therapeutic regimens for inhibiting breast cancer with aberrant Akt activity are essential. Here, we evaluated antitumor effect of a marine bacteria derived lipopeptide 'Iturin A' on human breast cancer in vitro and in vivo through disrupting Akt pathway. Proliferation of MDA-MB-231 and MCF-7 breast cancer cells were significantly inhibited by Iturin A and it induced apoptosis as confirmed by increased Sub G1 populations, DNA fragmentation, morphological changes and western blot analysis. Furthermore, Iturin A inhibited EGF induced Akt phosphorylation (Ser473 and Thr308) and its downstream targets GSK3β and FoxO3a. Iturin A inactivated MAPK as well as Akt kinase leading to the translocation of FoxO3a to the nucleus. Gene silencing of Akt in MDA-MB-231 and MCF-7 cells reduced the sensitivity of cancer cells to Iturin A. Interestingly, overexpression of Akt with Akt plasmid in cancer cells caused highly susceptible to induce apoptosis by Iturin A treatment. In a xenograft model, Iturin A inhibited tumor growth with reduced expressions of Ki-67, CD-31, P-Akt, P-GSK3β, P-FoxO3a and P-MAPK. Collectively, these findings imply that Iturin A has potential anticancer effect on breast cancer. PMID:25974307

  8. Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Xu; Li-Jie Zhao; Yan Li

    2009-01-01

    AIM: To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action. METHODS: The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential (ΔΨm) were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases (PI3K). RESULTS: Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 μmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt. CONCLUSION: Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.

  9. SCGB3A2 Inhibits Acrolein-Induced Apoptosis through Decreased p53 Phosphorylation

    International Nuclear Information System (INIS)

    Chronic obstructive pulmonary disease (COPD), a major global health problem with increasing morbidity and mortality rates, is anticipated to become the third leading cause of death worldwide by 2020. COPD arises from exposure to cigarette smoke. Acrolein, which is contained in cigarette smoke, is the most important risk factor for COPD. It causes lung injury through altering apoptosis and causes inflammation by augmenting p53 phosphorylation and producing reactive oxygen species (ROS). Secretoglobin (SCGB) 3A2, a secretory protein predominantly present in the epithelial cells of the lungs and trachea, is a cytokine-like small molecule having anti-inflammatory, antifibrotic, and growth factor activities. In this study, the effect of SCGB3A2 on acrolein-related apoptosis was investigated using the mouse fibroblast cell line MLg as the first step in determining the possible therapeutic value of SCGB3A2 in COPD. Acrolein increased the production of ROS and phosphorylation of p53 and induced apoptosis in MLg cells. While the extent of ROS production induced by acrolein was not affected by SCGB3A2, p53 phosphorylation was significantly decreased by SCGB3A2. These results demonstrate that SCGB3A2 inhibited acrolein-induced apoptosis through decreased p53 phosphorylation, not altered ROS levels

  10. Fe65 Is Phosphorylated on Ser289 after UV-Induced DNA Damage

    Science.gov (United States)

    Langlands, Hannah; Blain, Peter G.; Jowsey, Paul A.

    2016-01-01

    Fe65 undergoes a phosphatase-sensitive gel mobility shift after DNA damage, consistent with protein phosphorylation. A recent study identified Ser228 as a specific site of phosphorylation, targeted by the ATM and ATR protein kinases, with phosphorylation inhibiting the Fe65-dependent transcriptional activity of the amyloid precursor protein (APP). The direct binding of Fe65 to APP not only regulates target gene expression, but also contributes to secretase-mediated processing of APP, producing cytoactive proteolytic fragments including the APP intracellular domain (AICD) and cytotoxic amyloid β (Aβ) peptides. Given that the accumulation of Aβ peptides in neural plaques is a pathological feature of Alzheimer’s disease (AD), it is essential to understand the mechanisms controlling Aβ production. This will aid in the development of potential therapeutic agents that act to limit the deleterious production of Aβ peptides. The Fe65-APP complex has transcriptional activity and the complex is regulated by multiple post-translational modifications and other protein binding partners. In the present study, we have identified Ser289 as a novel site of UV-induced phosphorylation. Interestingly, this phosphorylation was mediated by ATM, rather than ATR, and occurred independently of APP. Neither phosphorylation nor mutation of Ser289 affected the Fe65-APP interaction, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Using mutagenesis, we demonstrated that Fe65 Ser289 phosphorylation did not affect the transcriptional activity of the Fe65-APP complex, in contrast to the previously described Ser228 site. PMID:27176072

  11. Phosphorylation of NMDA NR1 subunits in the myenteric plexus during TNBS induced colitis.

    Science.gov (United States)

    Zhou, QiQi; Caudle, Robert M; Moshiree, Baharak; Price, Donald D; Verne, G Nicholas

    2006-10-01

    N-Methyl-d-aspartic acid (NMDA) receptors are known to function in the mediation of pain and have a significant role in the development of hyperalgesia following inflammation. Serine phosphorylation regulation of NMDA receptor function occurs in a variety of conditions. No studies have demonstrated a change in phosphorylation of enteric NMDA receptors following colonic inflammation. We examined the levels of NMDA NR1 phosphorylation in trinitrobenzene sulfonic acid (TNBS) induced colitis in rats and compared it to protein translation and the development of visceral hypersensitivity. We have previously, demonstrated an increase in the C1 cassette of NR1 mRNA expression at 14, 21, and 28 days following TNBS administration. In this study, we examined the NR1 serine phosphorylation at 14 days following TNBS injection. Male Sprague-Dawley rats (200-250 g) were treated with TNBS (20mg per rat) diluted in 50% ethanol (n=3) and vehicle controls of 50% ethanol (n=3). TNBS and vehicle controls were administered with a 24 gauge catheter inserted into the lumen of the rat colon. The animals were sacrificed at 14 days after induction of the colitis and their distal colon was retrieved for two-dimensional (2D) western blot analysis. Serine phosphorylation of the NR1 subunit with C1 cassette appears at 14 days after TNBS injection. In contrast, there was no NR1-C1 expression in the vehicle controls and untreated normal controls. These results suggest a role for colonic-NMDA receptor phosphorylation in the development of neuronal plasticity following colonic inflammation. Phosphorylation of NR1 may partially explain visceral hypersensitivity present during colonic inflammation. PMID:16942839

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

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Bin; Lee, Eun Byul; Cui, Jun; Kim, Yosup [Department of Molecular Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 406-799 (Korea, Republic of); Jang, Ho Hee, E-mail: hhjang@gachon.ac.kr [Department of Molecular Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 406-799 (Korea, Republic of); Gachon Medical Research Institute, Gil Medical Center, Gachon University, Incheon (Korea, Republic of)

    2015-03-06

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

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

    International Nuclear Information System (INIS)

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

  14. Roles of the PI3K/Akt pathway in Epstein-Barr virus-induced cancers and therapeutic implications.

    Science.gov (United States)

    Chen, Jiezhong

    2012-12-12

    Viruses have been shown to be responsible for 10%-15% of cancer cases. Epstein-Barr virus (EBV) is the first virus to be associated with human malignancies. EBV can cause many cancers, including Burkett's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma and gastric cancer. Evidence shows that phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) plays a key role in EBV-induced malignancies. The main EBV oncoproteins latent membrane proteins (LMP) 1 and LMP2A can activate the PI3K/Akt pathway, which, in turn, affects cell survival, apoptosis, proliferation and genomic instability via its downstream target proteins to cause cancer. It has also been demonstrated that the activation of the PI3K/Akt pathway can result in drug resistance to chemotherapy. Thus, the inhibition of this pathway can increase the therapeutic efficacy of EBV-associated cancers. For example, PI3K inhibitor Ly294002 has been shown to increase the effect of 5-fluorouracil in an EBV-associated gastric cancer cell line. At present, dual inhibitors of PI3K and its downstream target mammalian target of rapamycin have been used in clinical trials and may be included in treatment regimens for EBV-associated cancers. PMID:24175221

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

    Science.gov (United States)

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

    2015-09-01

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

  16. The PI3K-Akt-mTOR pathway regulates Aβ oligomer induced neuronal cell cycle events

    Directory of Open Access Journals (Sweden)

    Herrup Karl

    2009-03-01

    Full Text Available Abstract Accumulating evidence suggests that neurons prone to degeneration in Alzheimer's Disease (AD exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP mouse model of AD (R1.40, neuronal cell cycle events (CCEs occur in the absence of beta-amyloid (Aβ deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP. These data suggested that soluble Aβ species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Aβ oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Aβ monomer or Aβ fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Aβ oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin and that PI3K, Akt or mTOR inhibitors blocked Aβ oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Aβ oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

  17. Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

    International Nuclear Information System (INIS)

    Objective: To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM. Methods: Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy. The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively. The micronucleus(MN)was analyzed by CB method to evaluate DNA damage. Results: FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses, and the fitting curve was shown as Y=3.96+11.29D-0.45D2. The level of phosphorylated ATM (p-ATM) was not changed significantly by using the same method. Western blot showed that p-ATM protein expression was significantly increased after irradiation compared with sham, irradiated group. The MN assay which represented DNA damage was sensitive to different doses. Conclusions: γ-ray irradiation could induce the phosphorylation of H2AX and ATM, which may play an important role in indicating DNA damage. Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage. (authors)

  18. Involvement of Protein Phosphorylation in Water Stress-induced Antioxidant Defense in Maize Leaves

    Institute of Scientific and Technical Information of China (English)

    Shu-cheng Xu; Hai-dong Ding; Feng-xia Su; A-ying Zhang; Ming-yi Jiang

    2009-01-01

    Using pharmacological and biochemical approaches, the role of protein phosphorylation and the interrelationship between water stress-enhanced kinase activity, antioxidant enzyme activity, hydrogen peroxide (H2O2) accumulation and endogenous abscisic acid in maize (Zea mays L.) leaves were investigated. Water-stress upregulated the activities of total protein phosphorylation and Ca2+ -dependent protein kinase, and the upregulation was blocked in abscisic acid-deficient vp5 mutant. Furthermore, pretreatments with a nicotinamide adenine dinucleotide phosphate oxidase inhibitor and a scavenger of H2O2 significantly reduced the increased activities of total protein kinase and Ca2+-dependent protein kinase in maize leaves exposed to water stress. Pretreatments with different protein kinase inhibitors also reduced the water stress-induced H2O2 production and the water stress-enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The data suggest that protein phosphorylation and H2O2 generation are required for water stress-induced antioxidant defense in maize leaves and that crosstalk between protein phosphorylation and H2O2 generation may occur.

  19. 4-Hydroxy estradiol but not 2-hydroxy estradiol induces expression of hypoxia-inducible factor 1α and vascular endothelial growth factor A through phosphatidylinositol 3-kinase/Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells

    International Nuclear Information System (INIS)

    Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. HIF-1 activates downstream target genes such as vascular endothelial growth factor A (VEGF-A), which plays an important role in tumor progression and angiogenesis. Estrogen exposure is considered to be the major risk factor for ovarian cancer. Estradiol (E2) is usually metabolized by CYP1A1/1A2 and CYP3A4 to the 2-hydroxy estradiol (2-OHE2) and 4-hydroxy estradiol (4-OHE2) in human liver. Many reports have suggested that the formation of 4-OHE2 is important for mammary carcinogenesis. However, the formation of 2-OHE2 may play an important role in exhibiting anticarcinogenic effects. In the present study, we have demonstrated that one of the catechol estrogen metabolites of E2, 4-OHE2, induces HIF-1α and VEGF-A expression at protein level in two human ovarian cancer cell lines, OVCAR-3 and A2780-CP70 cells, in dose- and time-dependent manners, whereas the other catechol estrogen metabolite of E2, 2-OHE2, does not alter HIF-1α and VEGF-A expression. To explore the mechanism of 4-OHE2-induced HIF-1α and VEGF-A expression, we studied whether phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) signaling pathways are involved in 4-OHE2-induced HIF-1α and VEGF-A expression. Our findings indicate that PI3K inhibitors, LY294002 and wortmannin, inhibited HIF-1α and VEGF-A expression, whereas MAPK inhibitor, PD98059, did not alter HIF-1α and VEGF-A expression induced by 4-OHE2. 4-OHE2, but not 2-OHE2, also induced Akt phosphorylation at Ser473 in dose- and time-dependent manners, and LY294002 and wortmannin inhibited Akt phosphorylation at Ser473 induced by 4-OHE2. Our results also indicated that the mTOR/FRAP inhibitor, rapamycin, inhibited 4-OHE2-induced HIF-1α and VEGF-A expression. These results suggest that the PI3K/Akt

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Impaired insulin-induced site-specific phosphorylation of TBC1 domain family, member 4 (TBC1D4) in skeletal muscle of type 2 diabetes patients is restored by endurance exercise-training

    DEFF Research Database (Denmark)

    Vind, B. F.; Pehmøller, Christian; Treebak, Jonas Thue; Birk, Jesper Bratz; Hey-Mogensen, M.; Beck-Nielsen, H.; Zierath, J. R.; Wojtaszewski, Jørgen; Højlund, K.

    2011-01-01

    -activated kinase (AMPK), glycogen synthase, hexokinase II and GLUT4 (20-75%). CONCLUSIONS/INTERPRETATION: Impaired insulin-induced site-specific TBC1D4 phosphorylation may contribute to skeletal muscle insulin resistance in type 2 diabetes. The mechanisms by which exercise-training improves insulin sensitivity in...... type 2 diabetes may involve augmented signalling of TBC1D4 and increased skeletal muscle content of key insulin signalling and effector proteins, e.g., Akt, TBC1D4, AMPK, glycogen synthase, GLUT4 and hexokinase II.......AIMS/HYPOTHESIS: Insulin-mediated glucose disposal rates (R (d)) are reduced in type 2 diabetic patients, a process in which intrinsic signalling defects are thought to be involved. Phosphorylation of TBC1 domain family, member 4 (TBC1D4) is at present the most distal insulin receptor signalling...

  2. Marine Cyclotripeptide X-13 Promotes Angiogenesis in Zebrafish and Human Endothelial Cells via PI3K/Akt/eNOS Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Zhong Pei

    2012-06-01

    Full Text Available Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508. We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or l-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.

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

    International Nuclear Information System (INIS)

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin β1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin β1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin β1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function

  4. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    Science.gov (United States)

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  5. Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

    OpenAIRE

    Zhao Zhang; Yun Zhang; Zheng Mou; Shifeng Chu; Xiaoyu Chen; Wenbin He; Xiaofeng Guo; Yuhe Yuan; Masami Takahashi; Naihong Chen

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and ce...

  6. Factor XII stimulates ERK1/2 and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis

    Science.gov (United States)

    LaRusch, Gretchen A.; Mahdi, Fakhri; Shariat-Madar, Zia; Adams, Gregory; Sitrin, Robert G.; Zhang, Wan Ming; McCrae, Keith R.

    2010-01-01

    Factor XII (FXII) and high molecular weight kininogen (HK) mutually block each other's binding to the urokinase plasminogen activator receptor (uPAR). We investigated if FXII stimulates cells by interacting with uPAR. FXII (3-62nM) with 0.05mM Zn2+ induces extracellular signal-related kinase 1/2 (ERK1/2; mitogen-activated protein kinase 44 [MAPK44] andMAPK42) and Akt (Ser473) phosphorylation in endothelial cells. FXII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. An uPAR signaling region for FXII is on domain 2 adjacent to uPAR's integrin binding site. Cleaved HK or peptides from HK's domain 5 blocks FXII-induced ERK1/2 and Akt phosphorylation. A β1 integrin peptide that binds uPAR, antibody 6S6 to β1 integrin, or the epidermal growth factor receptor (EGFR) inhibitor AG1478 blocks FXII-induced phosphorylation of ERK1/2 and Akt. FXII induces endothelial cell proliferation and 5-bromo-2′deoxy-uridine incorporation. FXII stimulates aortic sprouting in normal but not uPAR-deficient mouse aorta. FXII produces angiogenesis in matrigel plugs in normal but not uPAR-deficient mice. FXII knockout mice have reduced constitutive and wound-induced blood vessel number. In sum, FXII initiates signaling mediated by uPAR, β1 integrin, and the EGFR to induce human umbilical vein endothelial cell proliferation, growth, and angiogenesis. PMID:20228268

  7. MEK inhibition induces apoptosis in osteosarcoma cells with constitutive ERK1/2 phosphorylation.

    Science.gov (United States)

    Baranski, Zuzanna; Booij, Tijmen H; Kuijjer, Marieke L; de Jong, Yvonne; Cleton-Jansen, Anne-Marie; Price, Leo S; van de Water, Bob; Bovée, Judith V M G; Hogendoorn, Pancras C W; Danen, Erik H J

    2015-11-01

    Conventional high-grade osteosarcoma is the most common primary bone cancer with relatively high incidence in young people. Recurrent and metastatic tumors are difficult to treat. We performed a kinase inhibitor screen in two osteosarcoma cell lines, which identified MEK1/2 inhibitors. These inhibitors were further validated in a panel of six osteosarcoma cell lines. Western blot analysis was performed to assess ERK activity and efficacy of MEK inhibition. A 3D culture system was used to validate results from 2D monolayer cultures. Gene expression analysis was performed to identify differentially expressed gene signatures in sensitive and resistant cell lines. Activation of the AKT signaling network was explored using Western blot and pharmacological inhibition. In the screen, Trametinib, AZD8330 and TAK-733 decreased cell viability by more than 50%. Validation in six osteosarcoma cell lines identified three cell lines as resistant and three as sensitive to the inhibitors. Western blot analysis of ERK activity revealed that sensitive lines had high constitutive ERK activity. Treatment with the three MEK inhibitors in a 3D culture system validated efficacy in inhibition of osteosarcoma viability. MEK1/2 inhibition represents a candidate treatment strategy for osteosarcomas displaying high MEK activity as determined by ERK phosphorylation status. PMID:26807203

  8. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  9. Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages.

    Science.gov (United States)

    Yeh, J L; Hsu, J H; Hong, Y S; Wu, J R; Liang, J C; Wu, B N; Chen, I J; Liou, S F

    2011-01-01

    Eugenol and isoeugenol, two components of clover oil, have been reported to possess several biomedical properties, such as anti-inflammatory, antimicrobial and antioxidant effects. This study aims to examine the anti-inflammatory effects of eugenol, isoeugenol and four of their derivatives on expression of inducible nitric oxide synthase (iNOS) activated by lipopolysaccharide (LPS) in mouse macrophages (RAW 264.7), and to investigate molecular mechanisms underlying these effects. We found that two derivatives, eugenolol and glyceryl-isoeugenol, had potent inhibitory effects on LPS-induced upregulation of nitrite levels, iNOS protein and iNOS mRNA. In addition, they both suppressed the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced by LPS. Moreover, they both attenuated the DNA binding of NF-kB and AP-1, phosphorylation of inhibitory kB-alpha (IkB-alpha), and nuclear translocation of p65 protein induced by LPS. Finally, we demonstrated that glyceryl-isoeugenol suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK, whereas eugenolol suppressed the phosphorylation of ERK1/2 and p38 MAPK. Taken together, these results suggest that that eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kB and AP-1 through inhibition of MAPKs and Akt/IkB-alpha signaling pathways. Thus, this study implies that eugenolol and glyceryl-isoeugenol may provide therapeutic benefits for inflammatory diseases. PMID:21658309

  10. Methane limit LPS-induced NF-κB/MAPKs signal in macrophages and suppress immune response in mice by enhancing PI3K/AKT/GSK-3β-mediated IL-10 expression

    Science.gov (United States)

    Zhang, Xu; Li, Na; Shao, Han; Meng, Yan; Wang, Liping; Wu, Qian; Yao, Ying; Li, Jinbao; Bian, Jinjun; Zhang, Yan; Deng, Xiaoming

    2016-01-01

    Inflammatory diseases such as sepsis and autoimmune colitis, characterized by an overwhelming activation of the immune system and the counteracting anti-inflammatory response, remain a major health problem in worldwide. Emerging evidence suggests that methane have a protective effect on many animal models, like ischaemia reperfusion injury and diabetes-associated diseases. Whether methane could modulating inflammatory diseases remains largely unknown. Here we show that methane-rich saline (MS) ip treatment (16 ml/kg) alleviated endotoxin shock, bacteria-induced sepsis and dextran-sulfate-sodium-induced colitis in mice via decreased production of TNF-α and IL-6. In MS-treated macrophages, LPS-induced activation of NF-κb/MAPKs was attenuated. Interestingly, MS treatment significantly elevated the levels of IL-10 both in vitro and in vivo. Neutralization of IL-10 abrogated the therapeutic effect of MS. Moreover, anti-IL10 blockade partially restored the MS-mediated attenuation of NF-κb/MAPKs phosphorylation. We further found that MS resulted in markedly enhanced phosphorylation of GSK-3β and AKT, which both mediate the release of Il-10. Additionally, inhibition of PI3K attenuated MS-mediated p-GSK-3β and IL-10 production and reversed the suppressed activation of NF-κb/ MAPKs in response to LPS. Our results reveal a novel effect and mechanisms of methane and support the potential value of MS as a therapeutic approach in innate inflammatory diseases. PMID:27405597

  11. In vivo treatment with diphenyl ditelluride induces neurodegeneration in striatum of young rats: Implications of MAPK and Akt pathways

    Energy Technology Data Exchange (ETDEWEB)

    Heimfarth, Luana; Loureiro, Samanta Oliveira; Dutra, Márcio Ferreira; Andrade, Cláudia; Pettenuzzo, Letícia; Guma, Fátima T. Costa Rodrigues; Gonçalves, Carlos Alberto Saraiva [Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS (Brazil); Batista Teixeira da Rocha, João [Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS Brazil (Brazil); Pessoa-Pureur, Regina, E-mail: rpureur@ufrgs.br [Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS (Brazil)

    2012-10-15

    In the present report 15 day-old Wistar rats were injected with 0.3 μmol of diphenyl ditelluride (PhTe){sub 2}/kg body weight and parameters of neurodegeneration were analyzed in slices from striatum 6 days afterwards. We found hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein—GFAP and vimentin) and from neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H, respectively) and increased MAPK (Erk, JNK and p38MAPK) as well as PKA activities. The treatment induced reactive astrogliosis in the striatum, evidenced by increased GFAP and vimentin immunocontent as well as their mRNA overexpression. Also, (PhTe){sub 2} significantly increased the propidium iodide (PI) positive cells in NeuN positive population without altering PI incorporation into GFAP positive cells, indicating that in vivo exposure to (PhTe){sub 2} provoked neuronal damage. Immunohistochemistry showed a dramatic increase of GFAP staining characteristic of reactive astrogliosis. Moreover, increased caspase 3 in (PhTe){sub 2} treated striatal slices suggested apoptotic cell death. (PhTe){sub 2} exposure decreased Akt immunoreactivity, however phospho-GSK-3-β (Ser9) was unaltered, suggesting that this kinase is not directly implicated in the neurotoxicity of this compound. Therefore, the present results shed light into the mechanisms of (PhTe){sub 2}-induced neurodegeneration in rat striatum, evidencing a critical role for the MAPK and Akt signaling pathways and disruption of cytoskeletal homeostasis, which could be related with apoptotic neuronal death and astrogliosis. -- Highlights: ► Diphenyl ditelluride causes apoptotic neuronal death in the striatum of young rats. ► Diphenyl ditelluride causes reactive astrogliosis in the striatum of rats. ► Diphenyl ditelluride disrupts the homeostasis of the cytoskeleton of the striatum. ► The actions of diphenyl ditelluride are mediated by MAPK and Akt

  12. Escitalopram attenuates β-amyloid-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathway.

    Science.gov (United States)

    Wang, Yan-Juan; Ren, Qing-Guo; Gong, Wei-Gang; Wu, Di; Tang, Xiang; Li, Xiao-Li; Wu, Fang-Fang; Bai, Feng; Xu, Lin; Zhang, Zhi-Jun

    2016-03-22

    Tau hyperphosphorylation is an important pathological feature of Alzheimer's disease (AD). To investigate whether escitalopram could inhibit amyloid-β (Aβ)-induced tau hyperphosphorylation and the underlying mechanisms, we treated the rat primary hippocampal neurons with Aβ1-42 and examined the effect of escitalopram on tau hyperphosphorylation. Results showed that escitalopram decreased Aβ1-42-induced tau hyperphosphorylation. In addition, escitalopram activated the Akt/GSK-3β pathway, and the PI3K inhibitor LY294002 blocked the attenuation of tau hyperphosphorylation induced by escitalopram. Moreover, the 5-HT1A receptor agonist 8-OH-DPAT also activated the Akt/GSK-3β pathway and decreased Aβ1-42-induced tau hyperphosphorylation. Furthermore, the 5-HT1A receptor antagonist WAY-100635 blocked the activation of Akt/GSK-3β pathway and the attenuation of tau hyperphosphorylation induced by escitalopram. Finally, escitalopram improved Aβ1-42 induced impairment of neurite outgrowth and spine density, and reversed Aβ1-42 induced reduction of synaptic proteins. Our results demonstrated that escitalopram attenuated Aβ1-42-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathway. PMID:26950279

  13. Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis via the PI3K/AKT and ERK1/2 MAPK Pathways.

    Science.gov (United States)

    Lim, Whasun; Park, Sunwoo; Bazer, Fuller W; Song, Gwonhwa

    2016-12-01

    Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat, and sprouts. It has a variety of biological properties including anti-oxidant, anti-mutagenic, anti-carcinogenic, anti-inflammatory, anti-proliferative, and anti-spasmodic effects. Based on epidemiological and case-control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti-cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK, and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose-dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti-proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. J. Cell. Physiol. 231: 2690-2699, 2016. © 2016 Wiley Periodicals, Inc. PMID:26970256

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

    International Nuclear Information System (INIS)

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

  15. KMUP-1 Attenuates Endothelin-1-Induced Cardiomyocyte Hypertrophy through Activation of Heme Oxygenase-1 and Suppression of the Akt/GSK-3β, Calcineurin/NFATc4 and RhoA/ROCK Pathways

    Directory of Open Access Journals (Sweden)

    Shu-Fen Liou

    2015-06-01

    Full Text Available The signaling cascades of the mitogen activated protein kinase (MAPK family, calcineurin/NFATc4, and PI3K/Akt/GSK3, are believed to participate in endothelin-1 (ET-1-induced cardiac hypertrophy. The aim of this study was to investigate whether KMUP-1, a synthetic xanthine-based derivative, prevents cardiomyocyte hypertrophy induced by ET-1 and to elucidate the underlying mechanisms. We found that in H9c2 cardiomyocytes, stimulation with ET-1 (100 nM for 4 days induced cell hypertrophy and enhanced expressions of hypertrophic markers, including atrial natriuretic peptide and brain natriuretic peptide, which were all inhibited by KMUP-1 in a dose-dependent manner. In addition, KMUP-1 prevented ET-1-induced intracellular reactive oxygen species generation determined by the DCFH-DA assay in cardiomyocytes. KMUP-1 also attenuated phosphorylation of ERK1/2 and Akt/GSK-3β, and activation of calcineurin/NFATc4 and RhoA/ROCK pathways induced by ET-1. Furthermore, we found that the expression of heme oxygenase-1 (HO-1, a stress-response enzyme implicated in cardio-protection, was up-regulated by KMUP-1. Finally, KMUP-1 attenuated ET-1-stimulated activator protein-1 DNA binding activity. In conclusion, KMUP-1 attenuates cardiomyocyte hypertrophy induced by ET-1 through inhibiting ERK1/2, calcineurin/NFATc4 and RhoA/ROCK pathways, with associated cardioprotective effects via HO-1 activation. Therefore, KMUP-1 may have a role in pharmacological therapy of cardiac hypertrophy.

  16. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    Science.gov (United States)

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  17. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    Science.gov (United States)

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug. PMID:26366782

  18. Arsenic trioxide overcomes rapamycin-induced feedback activation of AKT and ERK signaling to enhance the anti-tumor effects in breast cancer.

    Directory of Open Access Journals (Sweden)

    Cynthia Guilbert

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

  19. MeCP2 phosphorylation limits psychostimulant-induced behavioral and neuronal plasticity.

    Science.gov (United States)

    Deng, Jie V; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C; Greenberg, Michael E; Kenny, Paul J; Calakos, Nicole; West, Anne E

    2014-03-26

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors. PMID:24671997

  20. Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK–MAPK pathway

    OpenAIRE

    Ke, Qingdong; Li, Qin; Ellen, Thomas P.; Sun, Hong; Costa, Max

    2008-01-01

    Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated th...

  1. Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose

    Institute of Scientific and Technical Information of China (English)

    Yun-peng ZHAI; Qian LU; Yao-wu LIU; Qian CHENG; Ya-qin WEI; Fan ZHANG; Cheng-lin LI

    2013-01-01

    Aim:To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.Methods:Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H2O2 (10 μmol/L) for 16 h.NO release was quantified using the Griess assay.The TGF-β1 level was measured using ELISA.The protein expression of p-Akt,t-Akt,Bim,and iNOS was examined by Western blotting.The mRNA levels of TGF-β1 and Bim were measured using RT-PCR.The cell proliferation rate was estimated using a BrdU incorporation assay.Results:Treatment of the cells with HG,H2O2,or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI),TGF-β1 inhibitor SB431542,or PI3K inhibitor LY294002.Both HG and H2O2 significantly increased the protein and mRNA levels of TGF-β1 in the cells,and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI.Furthermore,the treatment with HG or H2O2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate,which was blocked by co-treatment with DPI,SB431542,or LY294002.Moreover,the treatment with HG or H2O2 significantly inhibited Bim protein and mRNA expression,which was reversed by co-treatment with DPI,SB431542,or LY294002.Conclusion:The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS,which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.

  2. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model.

    Science.gov (United States)

    Chang, Chih-Zen; Wu, Shu-Chuan; Chang, Chia-Mao; Lin, Chih-Lung; Kwan, Aij-Lie

    2015-01-01

    Upregulation of protein kinase B (PKB, also known as Akt) is observed within the cerebral arteries of subarachnoid hemorrhage (SAH) animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS) and Akt pathways in a SAH in vitro study. Basilar arteries (BAs) were obtained to examine phosphatidylinositol-3-kinase (PI3K), phospho-PI3K, Akt, phospho-Akt (Western blot) and morphological examination. Endothelins (ETs) and eNOS evaluation (Western blot and immunostaining) were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p Arctigenin (p Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH. PMID:26539501

  3. Tensin2 is a novel mediator in thrombopoietin (TPO)-induced cellular proliferation by promoting Akt signaling

    OpenAIRE

    Jung, Andre Scott; Kaushansky, Alexis; MacBeath, Gavin; Kaushansky, Kenneth

    2011-01-01

    Thrombopoietin (TPO) and its receptor c-Mpl are essential in the regulation of the hematopoietic stem and progenitors cells as well as for the differentiation of megakaryocytes into mature platelets. Once TPO binds to its receptor, an intracellular signaling process is initiated through Janus kinase (JAK-2)-induced phosphorylation of the c-Mpl intracellular domain. Although some protein mediators that transmit the effects of TPO have been identified, many remain undiscovered. Using an unbiase...

  4. Human urine extract CDA-2 induces apoptosis of myelodysplastic syndrome-derived MUTZ-1 cells through the PI3K/Akt signaling pathway in a caspase-3-dependent manner

    Institute of Scientific and Technical Information of China (English)

    Jian HUANG; Min YANG; Hui LIU; Jie JIN

    2008-01-01

    Aim: The aim of this study was to investigate the antitumoral activity of human urine extract against myelodysplastic syndrome (MDS)-derived MUTZ-1 cells in vitro and in vivo. Methods: The MDS-refractory anemia with excess of blasts (RAEB)-derived MUTZ-1 cell line was used to examine the effects of a human urine preparation, CDA-2, on the induction of growth arrest and apoptosis. Apoptotic proteins, including caspase family, Bcl-2 family, the inhibitor of apoptosis protein (IAP) family, and the F-LICE-like inhibitory protein (FLIP), as well as cell cycle-associated proteins were studied. The phosphoinositide 3 ki- nase (PI3K)/Akt survival signaling pathway and the NF-k B pathway were also examined. The caspase-3 inhibitor Z-DEVD-fmk was used to examine the involve- ment of caspase-3 and poly (ADP-ribose) polymerase (PARP). PI3K inhibitor LY294002 was used to examine the involvement of the PI3K/Akt signaling path- way in this apoptosis-inducing effect. MUTZ-1 cell xenografted serious com- bined immunodeficiency disease mice were used for the in vivo study. Results: We found that CDA-2 could induce growth arrest and apoptosis of MUTZ-l cells in vitro and in vivo. The main mechanisms were related to the inhibition of PI3Kp110or expression at the transcriptional level, which inactivated the phos- phorylation of Akt involving the prevention NF-KB phosphorylation and nuclear translocation, the downregulation of the IAP family and FLIPL protein, and the dephosphorylation of the Bad protein, which then triggered the activation of the caspase cascades. This phenomenon could be inhibited by the PI3K inhibitor LY294002 and caspase-3 inhibitor Z-DEVD-fmk. Conclusion: Our results demon- strate the presence of active components in the human urine extract that can induce the growth arrest and apoptosis of MDS-RAEB-derived MUTZ-1 cells and may involve the PI3K/Akt signaling pathway in a caspase-3-dependent manner. This may provide new insights for the treatment of high-risk MDS.

  5. Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line

    Institute of Scientific and Technical Information of China (English)

    Hong-zhen HU; Yue-bo YANG; Xiang-dong XU; Hong-wei SHEN; Yi-min SHU; Zi REN; Xiao-mao LI; Hui-ming SHEN; Hai-tao ZENG

    2007-01-01

    Aim:To investigate the apoptosis-inducing effect of oridonin,a diterpenoid isolated from Rabdosia rubescens,in the human cervical carcinoma HeLa cell line.Methods:A morphological analysis,nuclear condensation,and fragmentation of chromatin were monitored using Hoechst 33342 staining. Cell viability was assessed using the 3-(4,5-dimethylthiazol-(2)-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and the apoptosis-related activation in the HeLa cell line were evaluated by flow cytometry and Western blotting. Results:Oridonin suppressed the proliferation of the HeLa cell line in a dose- and time-dependent fashion. Oridonin treatment downregulated the activation of protein kinase B (Akt),the expression of forkhead box class O (FOXO) transcription factor,and glycogen synthase kinase 3 (GSK3). Oridonin also induced the release of cytochrome c accompanied by the activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase cleavage. In addition,Z-D(OMe)-E(OMe)-V-D(OMe)FMK (z-DEVD-fmk),an inhibitor of caspases,prevented caspase-3 activation and abrogated oridonin-induced cell death. Finally,oridonin treatment of the HeLa cell line downregulated the expression of the inhibitor of the apoptosis protein.Conclusion:Our results showed that oridonin-induced apoptosis involved several molecular pathways. Oridonin may suppress constitutively activated targets of phosphatidylinositol 3-kinase (Akt,FOXO,and GSK3) in the HeLa cell line,inhibiting the proliferation and induction of caspase-dependent apoptosis.

  6. Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

    Science.gov (United States)

    Eboli, M L; Mercanti, D; Ciotti, M T; Aquino, A; Castellani, L

    1994-10-01

    Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells. PMID:7891841

  7. Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation.

    Science.gov (United States)

    Zhou, Yan; Ruan, Zheng; Zhou, Lili; Shu, Xugang; Sun, Xiaohong; Mi, Shumei; Yang, Yuhui; Yin, Yulong

    2016-01-22

    Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. PMID:26740181

  8. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    Directory of Open Access Journals (Sweden)

    Li J

    2015-02-01

    Full Text Available Jie Li,1 Chao Zhang,1 Hongchuan Jiang,1 Jiao Cheng21Department of General Surgery, 2Department of Gynaecology and Obstetrics, Beijing Chao-Yang Hospital, Beijing, People’s Republic of ChinaAbstract: Hypoxia-inducible factor-1 (HIF-1 is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10-7 mol/L, by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future.Keywords: Andrographolide (Andro, HIF-1α, inhibit, breast cancer, hypoxia, PI3k/AKT/mTOR pathway

  9. Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis.

    Science.gov (United States)

    Kohorn, Bruce D; Hoon, Divya; Minkoff, Benjamin B; Sussman, Michael R; Kohorn, Susan L

    2016-04-01

    The wall-associated kinases (WAKs)(1)are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGsin planta Using N(14/)N(15)isotopicin vivometabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominantWAKallele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase

  10. MeCP2 Phosphorylation Limits Psychostimulant-Induced Behavioral and Neuronal Plasticity

    OpenAIRE

    Deng, Jie V; Wan, Yehong; Wang, Xiaoting; Cohen, Sonia; Wetsel, William C.; Greenberg, Michael E.; Kenny, Paul J.; Calakos, Nicole; West, Anne E.

    2014-01-01

    The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the r...

  11. Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling

    OpenAIRE

    Helmer, Rebecca A.; Panchoo, Marlyn; Dertien, Janet S.; Bhakta, Suhani M.; Hewetson, Aveline; Beverly S Chilton

    2010-01-01

    Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-i...

  12. Mineral induced phosphorylation of glycolate ion--a metaphor in chemical evolution

    Science.gov (United States)

    Kolb, V.; Zhang, S.; Xu, Y.; Arrhenius, G.

    1997-01-01

    Bilateral surface-active minerals with excess positive charge concentrate glycolate and trimetaphosphate ion from l0(-3) m aqueous solution to half-saturation of the internal surface sites, and induce phosphorylation of glycolate ion in the mineral with trimetaphosphate, sorbed from l0(-2) m solution. By utilizing reactants from dilute solution at near-neutral pH, and eliminating the need for participating organic nitrogen compounds, the reaction comprises several elements considered necessary for geochemical realism in models for molecular evolution.

  13. PKA-mediated phosphorylation of ATR promotes recruitment of XPA to UV-induced DNA damage.

    Science.gov (United States)

    Jarrett, Stuart G; Wolf Horrell, Erin M; Christian, Perry A; Vanover, Jillian C; Boulanger, Mary C; Zou, Yue; D'Orazio, John A

    2014-06-19

    The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals. PMID:24950377

  14. High glucose induces autophagy of MC3T3-E1 cells via ROS-AKT-mTOR axis.

    Science.gov (United States)

    Wang, Xiaoju; Feng, Zhengping; Li, Jiling; Chen, Lixue; Tang, Weixue

    2016-07-01

    In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis

  15. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Kazutoyo Yoda

    2012-06-01

    Full Text Available Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

  16. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

    OpenAIRE

    Yoda, Kazutoyo; He, Fang; Miyazawa, Kenji; Hiramatsu, Masaru

    2012-01-01

    Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.Key...

  17. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. PMID:26921637

  18. Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

    International Nuclear Information System (INIS)

    Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3β (GSK-3β). Depletion of endogenous GSK-3β by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3β and found that DIF-1 dephosphorylated GSK-3β on Ser9 and induced the nuclear translocation of GSK-3β, suggesting that DIF-1 activated GSK-3β. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3β-mediated phosphorylation of Thr286

  19. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

    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.

  20. TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    International Nuclear Information System (INIS)

    Highlights: ► TNF-α induces MMP-9 expression and secretion to promote RPE cell migration. ► MAPK activation is not critical for TNF-α-induced MMP-9 expression. ► Akt and mTORC1 signaling mediate TNF-α-induced MMP-9 expression. ► SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-α. -- Abstract: Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α 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-α-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-α-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-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  1. Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion-Induced Kidney Growth.

    Science.gov (United States)

    Wu, Huijuan; Chen, Jianchun; Xu, Jinxian; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2016-04-01

    The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in theseTsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast,Tsc1single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules ofTsc1andrpS6double-mutant mice. Furthermore, pharmacologic treatment ofTsc1single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis inTsc1-deleted kidneys. PMID:26296742

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Changjiang [MOE Key Lab of Environment and Health, Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Key Lab of Birth Defects and Reproductive Health of National Health and Family Planning Commission, Chongqing Population and Family Planning Science and Technology Research Institute, Chongqing 400020 (China); Yang, Jixin [Department of Pediatric Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030 (China); Fu, Wenjuan; Qi, Suqin; Wang, Chenmin; Quan, Chao [MOE Key Lab of Environment and Health, Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Yang, Kedi, E-mail: yangkd@mails.tjmu.edu.cn [MOE Key Lab of Environment and Health, Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China)

    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.

  3. Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity

    OpenAIRE

    Harada, Hisashi; Quearry, Bonnie; Ruiz-Vela, Antonio; Korsmeyer, Stanley J.

    2004-01-01

    The “BH3-only” proapoptotic BCL-2 family members initiate the intrinsic apoptotic pathway. A small interfering RNA knockdown of BIM confirms this BH3-only member is important for the cytokine-mediated homeostasis of hematopoietic cells. We show here that the phosphorylation status of BIM controls its proapoptotic activity. IL-3, a hematopoietic survival factor, induces extracellular signal-regulated kinase/mitogen-activated protein kinase-mediated phosphorylation of BIM on three serine sites ...

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

    Science.gov (United States)

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

    2016-05-01

    Nrg1β is critically involved in cardiac development and also maintains function of the adult heart. Studies conducted in animal models showed that it improves cardiac performance under a range of pathological conditions, which led to its introduction in clinical trials to treat heart failure. Recent work also implicated Nrg1β in the regenerative potential of neonatal and adult hearts. The molecular mechanisms whereby Nrg1β acts in cardiac cells are still poorly understood. In the present study, we analyzed the effects of Nrg1β on glucose uptake in neonatal rat ventricular myocytes and investigated to what extent mTOR/Akt signaling pathways are implicated. We show that Nrg1β enhances glucose uptake in cardiomyocytes as efficiently as IGF-I and insulin. Nrg1β causes phosphorylation of ErbB2 and ErbB4 and rapidly induces the phosphorylation of FAK (Tyr(861)), Akt (Thr(308) and Ser(473)), and its effector AS160 (Thr(642)). Knockdown of ErbB2 or ErbB4 reduces Akt phosphorylation and blocks the glucose uptake. The Akt inhibitor VIII and the PI3K inhibitors LY-294002 and Byl-719 abolish Nrg1β-induced phosphorylation and glucose uptake. Finally, specific mTORC2 inactivation after knockdown of rictor blocks the Nrg1β-induced increases in Akt-p-Ser(473) but does not modify AS160-p-Thr(642) or the glucose uptake responses to Nrg1β. In conclusion, our study demonstrates that Nrg1β enhances glucose uptake in cardiomyocytes via ErbB2/ErbB4 heterodimers, PI3Kα, and Akt. Furthermore, although Nrg1β activates mTORC2, the resulting Akt-Ser(473) phosphorylation is not essential for glucose uptake induction. These new insights into pathways whereby Nrg1β regulates glucose uptake in cardiomyocytes may contribute to the understanding of its regenerative capacity and protective function in heart failure. PMID:26979522

  5. Dexamethasone (DEX induces Osmotic stress transcription factor 1 (Ostf1 through the Akt-GSK3β pathway in freshwater Japanese eel gill cell cultures

    Directory of Open Access Journals (Sweden)

    S. C. Chow

    2013-03-01

    Osmosensing and osmoregulatory processes undertaken in gills of euryhaline fish are coordinated by integrative actions of various signaling molecules/transcriptional factors. Considerable numbers of studies report the hyper- and hypo-osmoregulatory functions of fish gills, by illustrating the process of gill cell remodeling and the modulation of the expression of ion channels/transporters. Comparatively mechanistic information relayed from signal integration to transcriptional regulation in mediating gill cell functions has not yet been elucidated. In this study we demonstrate the functional links from cortisol stimulation, to Akt activation, to the expression of the transcriptional factor, Ostf1. Using the synthetic glucocorticoid receptor agonist, dexamethasone (DEX, Ostf1 expression is found to be activated via glucocorticoid receptor (GR and mediated by the Akt-GSK3β signaling pathway. Pharmacological experiments using kinase inhibitors reveal that the expression of Ostf1 is negatively regulated by Akt activation. The inhibition of PI3K or Akt activities, by the specific kinase inhibitors (wortmannin, LY294002 or SH6, stimulates Ostf1 expression, while a reduction of GSK3β activity by LiCl reduces Ostf1 expression. Collectively, our report for the first time indicates that DEX can induce Ostf1 via GR, with the involvement of the Akt-GSK3β signaling pathway in primary eel gill cell cultures. The data also suggest that Ostf1 may play different roles in gill cell survival during seawater acclimation.

  6. Acute phencyclidine treatment induces extensive and distinct protein phosphorylation in rat frontal cortex.

    Science.gov (United States)

    Palmowski, Pawel; Rogowska-Wrzesinska, Adelina; Williamson, James; Beck, Hans C; Mikkelsen, Jens D; Hansen, Henrik H; Jensen, Ole N

    2014-03-01

    Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate receptor antagonist, induces psychotomimetic effects in humans and animals. Administration of PCP to rodents is used as a preclinical model for schizophrenia; however, the molecular mechanisms underlying the symptoms remain largely unknown. Acute PCP treatment rapidly induces behavioral and cognitive deficits; therefore, post-translational regulation of protein activity is expected to play a role at early time points. We performed mass-spectrometry-driven quantitative analysis of rat frontal cortex 15, 30, or 240 min after the administration of PCP (10 mg/kg). We identified and quantified 23,548 peptides, including 4749 phosphopeptides, corresponding to 2604 proteins. A total of 352 proteins exhibited altered phosphorylation levels, indicating that protein phosphorylation is involved in the acute response to PCP. Computational assessment of the regulated proteins biological function revealed that PCP perturbs key processes in the frontal cortex including calcium homeostasis, organization of cytoskeleton, endo/exocytosis, and energy metabolism. This study on acute PCP treatment provides the largest proteomics and phosphoproteomics data sets to date of a preclinical model of schizophrenia. Our findings contribute to the understanding of alterations in glutamatergic neurotransmission in schizophrenia and provide a foundation for discovery of novel targets for pharmacological intervention. PMID:24564430

  7. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  8. Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana

    Science.gov (United States)

    Reymond, P.; Short, T. W.; Briggs, W. R.; Poff, K. L.

    1992-01-01

    Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

  9. mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue.

    Science.gov (United States)

    Albert, Verena; Svensson, Kristoffer; Shimobayashi, Mitsugu; Colombi, Marco; Muñoz, Sergio; Jimenez, Veronica; Handschin, Christoph; Bosch, Fatima; Hall, Michael N

    2016-03-01

    Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon β-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation. PMID:26772600

  10. Hedyotis diffusa plus Scutellaria barbata Induce Bladder Cancer Cell Apoptosis by Inhibiting Akt Signaling Pathway through Downregulating miR-155 Expression.

    Science.gov (United States)

    Pan, Li-Tao; Sheung, Yip; Guo, Wen-Peng; Rong, Zhi-Bin; Cai, Zhi-Ming

    2016-01-01

    Traditional Chinese medicine is increasingly used to treat cancer. Our clinical experiences identify Hedyotis diffusa plus Scutellaria barbata as the most common herb-pair (couplet medicinal) used for the core treatment of bladder cancer. This study aims to investigate the antitumor effect of the herb-pair in bladder cancer cells. The results show that Hedyotis diffusa plus Scutellaria barbata inhibited bladder cancer cell growth and clone formation in a dose-dependent and time-dependent manner. It also induced cell apoptosis through decreasing Akt activation and reducing the expression of antiapoptotic proteins Bcl-2 and Mcl-1. Further experiments showed that miR-155 was reduced by the herb-pair and miRNA-155 inhibitor induced cell apoptosis and suppressed Akt activation. Overexpression of miR-155 reversed herb-pair induced cell apoptosis through activating Akt pathway in both bladder cancer cell lines. The findings reveal that Hedyotis diffusa plus Scutellaria barbata reduce Akt activation through reducing miR-155 expression, resulting in cell apoptosis. It demonstrated the potential mechanism of Hedyotis diffusa plus Scutellaria barbata for the core treatment of bladder cancer. PMID:26989427

  11. Hedyotis diffusa plus Scutellaria barbata Induce Bladder Cancer Cell Apoptosis by Inhibiting Akt Signaling Pathway through Downregulating miR-155 Expression

    Directory of Open Access Journals (Sweden)

    Li-Tao Pan

    2016-01-01

    Full Text Available Traditional Chinese medicine is increasingly used to treat cancer. Our clinical experiences identify Hedyotis diffusa plus Scutellaria barbata as the most common herb-pair (couplet medicinal used for the core treatment of bladder cancer. This study aims to investigate the antitumor effect of the herb-pair in bladder cancer cells. The results show that Hedyotis diffusa plus Scutellaria barbata inhibited bladder cancer cell growth and clone formation in a dose-dependent and time-dependent manner. It also induced cell apoptosis through decreasing Akt activation and reducing the expression of antiapoptotic proteins Bcl-2 and Mcl-1. Further experiments showed that miR-155 was reduced by the herb-pair and miRNA-155 inhibitor induced cell apoptosis and suppressed Akt activation. Overexpression of miR-155 reversed herb-pair induced cell apoptosis through activating Akt pathway in both bladder cancer cell lines. The findings reveal that Hedyotis diffusa plus Scutellaria barbata reduce Akt activation through reducing miR-155 expression, resulting in cell apoptosis. It demonstrated the potential mechanism of Hedyotis diffusa plus Scutellaria barbata for the core treatment of bladder cancer.

  12. Piperlongumine, an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells.

    Science.gov (United States)

    Shrivastava, Shweta; Kulkarni, Prasad; Thummuri, Dinesh; Jeengar, Manish Kumar; Naidu, V G M; Alvala, Mallika; Redddy, G Bhanuprakash; Ramakrishna, Sistla

    2014-07-01

    The phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3 K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3 K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3 K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3 K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3 k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer. PMID:24729100

  13. 3-Bromopyruvate induces apoptosis in breast cancer cells by downregulating Mcl-1 through the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Liu, Zhe; Zhang, Yuan-Yuan; Zhang, Qian-Wen; Zhao, Su-Rong; Wu, Cheng-Zhu; Cheng, Xiu; Jiang, Chen-Chen; Jiang, Zhi-Wen; Liu, Hao

    2014-04-01

    The hexokinase inhibitor 3-bromopyruvate (3-BrPA) can inhibit glycolysis in tumor cells to reduce ATP production, resulting in apoptosis. However, as 3-BrPA is an alkylating agent, its cytotoxic action may be induced by other molecular mechanisms. The results presented here reveal that 3-BrPA-induced apoptosis is caspase independent. Further, 3-BrPA induces the generation of reactive oxygen species in MDA-MB-231 cells, leading to mitochondria-mediated apoptosis. These results suggest that caspase-independent apoptosis may be induced by the generation of reactive oxygen species. In this study, we also demonstrated that 3-BrPA induces apoptosis through the downregulation of myeloid cell leukemia-1 (Mcl-1) in MDA-MB-231 breast cancer cells. The results of Mcl-1 knockdown indicate that Mcl-1 plays an important role in 3-BrPA-induced apoptosis. Further, the upregulation of Mcl-1 expression in 3-BrPA-treated MDA-MB-231 cells significantly increases cell viability. In addition, 3-BrPA treatment resulted in the downregulation of p-Akt, suggesting that 3-BrPA may downregulate Mcl-1 through the phosphoinositide-3-kinase/Akt pathway. These findings indicate that 3-BrPA induces apoptosis in breast cancer cells by downregulating Mcl-1 through the phosphoinositide-3-kinase/Akt signaling pathway. PMID:24492287

  14. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

    OpenAIRE

    Nacerddine, Karim; Beaudry, Jean-Bernard; Ginjala, Vasudeva; Westerman, Bart; Mattiroli, Francesca; Song, Ji-Ying; Poel, Henk van der; Ponz, Olga Balagué; Pritchard, Colin; Cornelissen-Steijger, Paulien; Zevenhoven, John; Tanger, Ellen; Sixma, Titia K; Ganesan, Shridar; van Lohuizen, Maarten

    2012-01-01

    Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency....

  15. Pancreas-specific Pten deficiency causes partial resistance to diabetes and elevated hepatic AKT signaling

    Institute of Scientific and Technical Information of China (English)

    Zan Tong; Yan Fan; Weiqi Zhang; Jun Xu; Jing Cheng; Mingxiao Ding; Hongkui Deng

    2009-01-01

    PTEN, a negative regulator of the phosphatidylinositol-3-kinase/AKT pathway, is an important modulator of insu-lin signaling. To determine the metabolic function of pancreatic Pten, we generated pancreas-specific Pten knockout (PPKO) mice. PPKO mice had enlarged pancreas and elevated proliferation of acinar cells. They also exhibited hy-poglycemia, hypoinsulinemia, and altered amino metabolism. Notably, PPKO mice showed delayed onset of strepto-zotocin (STZ)-induced diabetes and sex-biased resistance to high-fat-diet (HFD)-induced diabetes. To investigate the mechanism for the resistance to HFD-induced hyperglycemia in PPKO mice, we evaluated AKT phosphorylation in major insulin-responsive tissues: the liver, muscle, and fat. We found that Pten loss in the pancreas causes the eleva-tion of AKT signaling in the liver. The phosphorylation of AKT and its downstream substrate GSK3β was increased in the liver of PPKO mice, while PTEN level was decreased without detectable excision of Pten allele in the liver of PPKO mice. Proteomics analysis revealed dramatically decreased level of 78-kDa glucose-regulated protein (GRP78) in the liver of PPKO mice, which may also contribute to the lower blood glucose level of PPKO mice fed with HFD. Together, our findings reveal a novel response in the liver to pancreatic defect in metabolic regulation, adding a new dimension to understanding diabetes resistance.

  16. Endostar, a novel recombinant human endostatin, exerts antiangiogenic effect via blocking VEGF-induced tyrosine phosphorylation of KDR/Flk-1 of endothelial cells

    International Nuclear Information System (INIS)

    Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence and forming another his-tag structure, was approved by the SFDA in 2005 for the treatment of non-small-cell lung cancer. But its mechanism of action has not been illustrated before. In this study, we examined the antiangiogenic activities of endostar in vitro and in vivo. The results showed that endostar suppressed the VEGF-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Endostar blocked microvessel sprouting from rat aortic rings in vitro. Moreover, it could inhibit the formation of new capillaries from pre-existing vessels in the chicken chorioallantoic membrane (CAM) assay and affect the growth of vessels in tumor. We further found the antiangiogenic effects of endostar were correlated with the VEGF-triggered signaling. Endostar suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1(VEGFR-2) as well as the overall VEGFR-2 expression and the activation of ERK, p38 MAPK, and AKT in HUVECs. Collectively, these data indicated the relationship between endostar and VEGF signal pathways and provided a molecular basis for the antiangiogenic effects of endostar

  17. PF573,228 inhibits vascular tumor cell growth, migration as well as angiogenesis, induces apoptosis and abrogates PRAS40 and S6RP phosphorylation.

    Science.gov (United States)

    Mabeta, Peace

    2016-09-01

    PF573,228 is a compound that targets focal adhesion kinase (FAK), a non-receptor protein kinase, which is over-expressed in various tumors. The aim of this study was to evaluate the effects of PF573,228 on the cells derived from mouse vascular tumors, namely, endothelioma cells. The treatment of endothelioma cells with PF573,228 reduced their growth with an IC50 of approximately 4.6 μmol L-1 and inhibited cell migration with an IC50 of about 0.01 μmol L-1. Microscopic studies revealed morphological attributes of apoptosis. These observations were confirmed by ELISA, which showed increased caspase-3 activity. PF573,228 also inhibited angiogenesis in a dose-dependent manner, with an IC50 of approximately 3.7 μmol L-1, and abrogated the phosphorylation of cell survival proteins, proline-rich Akt substrate (PRAS40) and S6 ribosomal protein (S6RP). Array data further revealed that PF573,228 induced caspase-3 activation, thus promoting apoptosis. Since all the processes inhibited by PF573,228 provide important support to tumor survival and progression, the drug may have a potential role in the treatment of vascular tumors. PMID:27383888

  18. Dual inhibition of CDK4/Rb and PI3K/AKT/mTOR pathways by ON123300 induces synthetic lethality in mantle cell lymphomas.

    Science.gov (United States)

    Divakar, S K A; Ramana Reddy, M V; Cosenza, S C; Baker, S J; Perumal, D; Antonelli, A C; Brody, J; Akula, B; Parekh, S; Reddy, E Premkumar

    2016-01-01

    This study describes the characterization of a novel kinase inhibitor, ON123300, which inhibits CDK4/6 (cyclin-dependent kinases 4 and 6) and phosphatidylinositol 3 kinase-δ (PI3K-δ) and exhibits potent activity against mantle cell lymphomas (MCLs) both in vitro and in vivo. We examined the effects of PD0332991 and ON123300 on cell cycle progression, modulation of the retinoblastoma (Rb) and PI3K/AKT pathways, and the induction of apoptosis in MCL cell lines and patient-derived samples. When Granta 519 and Z138C cells were incubated with PD0332991 and ON123300, both compounds were equally efficient in their ability to inhibit the phosphorylation of Rb family proteins. However, only ON123300 inhibited the phosphorylation of proteins associated with the PI3K/AKT pathway. Cells treated with PD0332991 rapidly accumulated in the G0/G1 phase of cell cycle as a function of increasing concentration. Although ON123300-treated cells arrested similarly at lower concentrations, higher concentrations resulted in the induction of apoptosis, which was not observed in PD0332991-treated samples. Mouse xenograft assays also showed a strong inhibition of MCL tumor growth in ON123300-treated animals. Finally, treatment of ibrutinib-sensitive and -resistant patient-derived MCLs with ON123300 also triggered apoptosis and inhibition of the Rb and PI3K/AKT pathways, suggesting that this compound might be an effective agent in MCL, including ibrutinib-resistant forms of the disease. PMID:26174628

  19. Tyrosine phosphorylation of a 66KD soluble protein and augmentation of lectin induced mitogenesis by DMSO in human T lymphocytes

    International Nuclear Information System (INIS)

    The authors have demonstrated that induction of mitogenesis in human T lymphocytes is associated with the tyrosine phosphorylation of a 66KD soluble substrate-TPP 66. Since DMSO has been shown to be a non-specific stimulator of tyrosine protein kinases they have examined the effect of DMSO on both activation and tyrosine phosphorylation in human T cells. Human peripheral blood T lymphocytes were isolated by dextran sedimentation, Ficol/Paque centrifugation and nylon wool filtration. Phosphorylation was performed in cells incubated with [32P] orthophosphate followed by DMSO for 30 min. TPP 66 was identified by 2-D PAGE, autoradiography, and HV electrophoresis of the hydrolyzed protein. Concentrations of DMSO from 1% to 50% induced the tyrosine phosphorylation of TPP 66 with maximal stimulation seen at 20%. DMSO alone did not activate the T cells (measured by [3H] thymidine incorporation) when tested at high concentrations for 30 sec to 10 min. (longer incubations were markedly toxic) or low concentrations for 12 to 48 hrs. Low concentrations of DMSO 0.1%-0.5% did however, markedly augment [3H] thymidine incorporation induced by PHA or Con A. These data suggest that tyrosine phosphorylation of TPP 66 alone may not constitute sufficient signal for the activation sequence to begin but the phosphorylation of this soluble substrate may be a critical factor in the propagation of the activation sequence

  20. Isoliquiritigenin induces growth inhibition and apoptosis through downregulating arachidonic acid metabolic network and the deactivation of PI3K/Akt in human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying; Zhao, Haixia [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Wang, Yuzhong [Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Zheng, Hao; Yu, Wei; Chai, Hongyan [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zhang, Jing [Animal Experimental Center of Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390,USA (United States); Guo, Austin M. [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Department of Pharmacology, New York Medical College, Valhalla, NY 10595 (United States); Yue, Jiang; Peng, Renxiu [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2013-10-01

    Arachidonic acid (AA)-derived eicosanoids and its downstream pathways have been demonstrated to play crucial roles in growth control of breast cancer. Here, we demonstrate that isoliquiritigenin, a flavonoid phytoestrogen from licorice, induces growth inhibition and apoptosis through downregulating multiple key enzymes in AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. Isoliquiritigenin diminished cell viability, 5-bromo-2′-deoxyuridine (BrdU) incorporation, and clonogenic ability in both MCF-7 and MDA-MB-231cells, and induced apoptosis as evidenced by an analysis of cytoplasmic histone-associated DNA fragmentation, flow cytometry and hoechst staining. Furthermore, isoliquiritigenin inhibited mRNA expression of multiple forms of AA-metabolizing enzymes, including phospholipase A2 (PLA2), cyclooxygenases (COX)-2 and cytochrome P450 (CYP) 4A, and decreased secretion of their products, including prostaglandin E{sub 2} (PGE{sub 2}) and 20-hydroxyeicosatetraenoic acid (20-HETE), without affecting COX-1, 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP), and leukotriene B{sub 4} (LTB{sub 4}). In addition, it downregulated the levels of phospho-PI3K, phospho-PDK (Ser{sup 241}), phospho-Akt (Thr{sup 308}), phospho-Bad (Ser{sup 136}), and Bcl-x{sub L} expression, thereby activating caspase cascades and eventually cleaving poly(ADP-ribose) polymerase (PARP). Conversely, the addition of exogenous eicosanoids, including PGE{sub 2}, LTB{sub 4} and a 20-HETE analog (WIT003), and caspase inhibitors, or overexpression of constitutively active Akt reversed isoliquiritigenin-induced apoptosis. Notably, isoliquiritigenin induced growth inhibition and apoptosis of MDA-MB-231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt (Thr{sup 308}). Collectively, these data suggest that isoliquiritigenin induces growth inhibition and apoptosis through downregulating AA metabolic

  1. Isoliquiritigenin induces growth inhibition and apoptosis through downregulating arachidonic acid metabolic network and the deactivation of PI3K/Akt in human breast cancer

    International Nuclear Information System (INIS)

    Arachidonic acid (AA)-derived eicosanoids and its downstream pathways have been demonstrated to play crucial roles in growth control of breast cancer. Here, we demonstrate that isoliquiritigenin, a flavonoid phytoestrogen from licorice, induces growth inhibition and apoptosis through downregulating multiple key enzymes in AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. Isoliquiritigenin diminished cell viability, 5-bromo-2′-deoxyuridine (BrdU) incorporation, and clonogenic ability in both MCF-7 and MDA-MB-231cells, and induced apoptosis as evidenced by an analysis of cytoplasmic histone-associated DNA fragmentation, flow cytometry and hoechst staining. Furthermore, isoliquiritigenin inhibited mRNA expression of multiple forms of AA-metabolizing enzymes, including phospholipase A2 (PLA2), cyclooxygenases (COX)-2 and cytochrome P450 (CYP) 4A, and decreased secretion of their products, including prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE), without affecting COX-1, 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP), and leukotriene B4 (LTB4). In addition, it downregulated the levels of phospho-PI3K, phospho-PDK (Ser241), phospho-Akt (Thr308), phospho-Bad (Ser136), and Bcl-xL expression, thereby activating caspase cascades and eventually cleaving poly(ADP-ribose) polymerase (PARP). Conversely, the addition of exogenous eicosanoids, including PGE2, LTB4 and a 20-HETE analog (WIT003), and caspase inhibitors, or overexpression of constitutively active Akt reversed isoliquiritigenin-induced apoptosis. Notably, isoliquiritigenin induced growth inhibition and apoptosis of MDA-MB-231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt (Thr308). Collectively, these data suggest that isoliquiritigenin induces growth inhibition and apoptosis through downregulating AA metabolic network and the deactivation of PI3K/Akt in human breast cancer

  2. Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27.

    Science.gov (United States)

    Hirose, H; Sakuma, N; Kaji, N; Nakayama, K; Inoue, K; Sekijima, M; Nojima, T; Miyakoshi, J

    2007-02-01

    An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family. PMID:17004241

  3. Ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of the HMGB1–RAGE and AKT pathways

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ping; Dai, Weiqi; Wang, Fan; Lu, Jie; Shen, Miao; Chen, Kan; Li, Jingjing; Zhang, Yan; Wang, Chengfen; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Guo, Chuan-Yong, E-mail: guochuanyong@hotmail.com; Xu, Ling, E-mail: xuling606@sina.com

    2014-01-24

    Highlights: • Ethyl pyruvate inhibits liver cancer. • Promotes apoptosis. • Decreased the expression of HMGB1, p-Akt. - Abstract: Ethyl pyruvate (EP) was recently identified as a stable lipophilic derivative of pyruvic acid with significant antineoplastic activities. The high mobility group box-B1 (HMGB1)–receptor for advanced glycation end-products (RAGE) and the protein kinase B (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. We tried to observe the effects of ethyl pyruvate on liver cancer growth and explored its effects in hepatocellular carcinoma model. In this study, three hepatocellular carcinoma cell lines were treated with ethyl pyruvate. An MTT colorimetric assay was used to assess the effects of EP on cell proliferation. Flow cytometry and TUNEL assays were used to analyze apoptosis. Real-time PCR, Western blotting and immunofluorescence demonstrated ethyl pyruvate reduced the HMGB1–RAGE and AKT pathways. The results of hepatoma orthotopic tumor model verified the antitumor effects of ethyl pyruvate in vivo. EP could induce apoptosis and slow the growth of liver cancer. Moreover, EP decreased the expression of HMGB1, RAGE, p-AKT and matrix metallopeptidase-9 (MMP9) and increased the Bax/Bcl-2 ratio. In conclusion, this study demonstrates that ethyl pyruvate induces apoptosis and cell-cycle arrest in G phase in hepatocellular carcinoma cells, plays a critical role in the treatment of cancer.

  4. HC toxin (a HDAC inhibitor) enhances IRS1-Akt signalling and metabolism in mouse myotubes.

    Science.gov (United States)

    Tan, Hayden Weng Siong; Sim, Arthur Yi Loong; Huang, Su Ling; Leng, Ying; Long, Yun Chau

    2015-12-01

    Exercise enhances numerous signalling pathways and activates substrate metabolism in skeletal muscle. Small molecule compounds that activate these cellular responses have been shown to recapitulate the metabolic benefits of exercise. In this study, a histone deacetylase (HDAC) inhibitor, HC toxin, was investigated as a small molecule compound that activates exercise-induced adaptations. In C2C12 myotubes, HC toxin treatment activated two exercise-stimulated pathways: AMP-activated protein kinase (AMPK) and Akt pathways. HC toxin increased the protein content and phosphorylation of insulin receptor substrate 1 as well as the activation of downstream Akt signalling. The effects of HC toxin on IRS1-Akt signalling were PI3K-dependent as wortmannin abolishes its effects on IRS1 protein accumulation and Akt phosphorylation. HC toxin-induced Akt activation was sufficient to enhance downstream mTOR complex 1 (mTORC1) signalling including p70S6K and S6, which were consistently abolished by PI3K inhibition. Insulin-stimulated glucose uptake, glycolysis, mitochondrial respiration and fatty acid oxidation were also enhanced in HC toxin-treated myotubes. When myotubes were challenged with serum starvation for the induction of atrophy, HC toxin treatment prevented the induction of genes that are involved in autophagy and proteasomal proteolysis. Conversely, IRS1-Akt signalling was not induced by HC toxin in several hepatoma cell lines, providing evidence for a favourable safety profile of this small molecule. These data highlight the potential of HDAC inhibitors as a novel class of small molecules for the induction of exercise-like signalling pathways and metabolism. PMID:26373795

  5. Nodularin Exposure Induces SOD1 Phosphorylation and Disrupts SOD1 Co-localization with Actin Filaments

    Directory of Open Access Journals (Sweden)

    Kari E. Fladmark

    2012-12-01

    Full Text Available Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS, which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1, without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized.

  6. Deprenyl prevents MPP+-induced oxidative damage in PC12 cells by the upregulation of Nrf2-mediated NQO1 expression through the activation of PI3K/Akt and Erk

    International Nuclear Information System (INIS)

    Highlights: ► We investigated deprenyl prevent MPP+-induced cytotoxicity in PC12 cells. ► Deprenyl increases NOQ1 expression by triggering Nrf2 nuclear translocation. ► PI3K/Akt and Erk signals were involved in Nrf2 translocation induced by deprenyl. ► Nrf2-mediated cytoprotective effect of deprenyl does not involve inhibition of MAO-B. -- Abstract: Neuroprotection has been the focus of several current efforts to develop a strategy for the treatment of Parkinson's disease (PD). The B-type monoamine oxidase (MAO-B) inhibitor deprenyl (selegiline) is used clinically as a PD therapeutic agent, however, its cytoprotective mechanism has not yet been fully elucidated. In this study, we show that deprenyl upregulates the expression and activity of NAD(P)H: quinone oxidoreductase 1 (NQO1), attenuates the increase in the quinoprotein levels in 1-methyl-4-phenylpyridinium (MPP+)-treated PC12 cells, and protects PC12 cells from oxidative damage. Deprenyl triggers the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway by increasing the nuclear translocation and DNA-binding activity of Nrf2. Both the antioxidant activity of deprenyl and its effect on NQO1 upregulation were greatly attenuated in Nrf2 siRNA transfected cells. The phosphorylation of extracellular regulating protein kinase (Erk) and Akt can be induced by the administration of 50 μM deprenyl in PC12 cells, and the ability of deprenyl to enhance NQO1 expression and Nrf2 nuclear translocation is partly attenuated by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and is almost completely attenuated by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002. The activation of Nrf2/ARE signaling by deprenyl in PC12 cells is independent of MAO-B inhibition. Altogether, our findings indicate that deprenyl protects PC12 cells exposed to MPP+ resulting from oxidative stress via the Nrf2-mediated upregulation of NQO1 involving both the PI3K/Akt and

  7. Break-induced replication requires DNA damage-induced phosphorylation of Pif1 and leads to telomere lengthening.

    Directory of Open Access Journals (Sweden)

    Yulia Vasianovich

    2014-10-01

    Full Text Available Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR. Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway - Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.

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

    International Nuclear Information System (INIS)

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

  9. Methylglyoxal induces platelet hyperaggregation and reduces thrombus stability by activating PKC and inhibiting PI3K/Akt pathway.

    Directory of Open Access Journals (Sweden)

    Karin Hadas

    Full Text Available Diabetes is characterized by a dysregulation of glucose homeostasis and platelets from patients with diabetes are known to be hyper-reactive and contribute to the accelerated development of vascular diseases. Since many of the deleterious effects of glucose have been attributed to its metabolite methylgyloxal (MG rather than to hyperglycemia itself, the aim of the present study was to characterize the effects of MG on platelet function. Washed human platelets were pre-incubated for 15 min with MG and platelet aggregation, adhesion on matrix-coated slides and signaling (Western blot were assessed ex vivo. In vivo, the effect of MG on thrombus formation was determined using the FeCl3-induced carotid artery injury model. MG potentiated thrombin-induced platelet aggregation and dense granule release, but inhibited platelet spreading on fibronectin and collagen. In vivo, MG accelerated thrombus formation but decreased thrombus stability. At the molecular level, MG increased intracellular Ca(2+ and activated classical PKCs at the same time as inhibiting PI3K/Akt and the β3-integrin outside-in signaling. In conclusion, these findings indicate that the enhanced MG concentration measured in diabetic patients can directly contribute to the platelet dysfunction associated with diabetes characterized by hyperaggregability and reduced thrombus stability.

  10. Reactive oxygen species mediate Cr(VI)-induced carcinogenesis through PI3K/AKT-dependent activation of GSK-3β/β-catenin signaling

    Energy Technology Data Exchange (ETDEWEB)

    Son, Young-Ok; Pratheeshkumar, Poyil; Wang, Lei; Wang, Xin; Fan, Jia; Kim, Dong-Hern; Lee, Ju-Yeon; Zhang, Zhuo [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); Lee, Jeong-Chae [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States); School of Dentistry and Institute of Oral Biosciences, Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin, E-mail: xshi5@email.uky.edu [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305 (United States)

    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

  11. Reactive oxygen species mediate Cr(VI)-induced carcinogenesis through PI3K/AKT-dependent activation of GSK-3β/β-catenin signaling

    International Nuclear Information System (INIS)

    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

  12. Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line

    International Nuclear Information System (INIS)

    The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C

  13. Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model

    Directory of Open Access Journals (Sweden)

    Chih-Zen Chang

    2015-01-01

    Full Text Available Upregulation of protein kinase B (PKB, also known as Akt is observed within the cerebral arteries of subarachnoid hemorrhage (SAH animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS and Akt pathways in a SAH in vitro study. Basilar arteries (BAs were obtained to examine phosphatidylinositol-3-kinase (PI3K, phospho-PI3K, Akt, phospho-Akt (Western blot and morphological examination. Endothelins (ETs and eNOS evaluation (Western blot and immunostaining were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p<0.01. The reduced eNOS protein and phospho-Akt expression in the SAH groups were relieved by the treatment of Arctigenin (p<0.01. This result confirmed that Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH.

  14. Tris(2-chloroethyl)phosphate-induced cell growth arrest via attenuation of SIRT1-independent PI3K/Akt/mTOR pathway.

    Science.gov (United States)

    Zhang, Wenjuan; Zhang, Youjian; Wang, Zhiyuan; Xu, Tian; Huang, Cheng; Yin, Wenjun; Wang, Jing; Xiong, Wei; Lu, Wenhong; Zheng, Hongyan; Yuan, Jing

    2016-07-01

    Tris(2-chloroethyl)phosphate (TCEP) as an organophosphorus flame retardant and plasticizer has been widely used in industrial and household products. It not only was detected in residential indoor air and dust, surface and drinking water, but also in human plasma and breast milk, and tissue samples of liver, kidneys and brain from rodents. TCEP is classified as carcinogenic category 2 and toxic for reproduction category 1B. Sufficient evidence from experimental animals indicated carcinogenicity of TCEP in the liver, and kidneys as well as cell loss in the brain. However, the underlying mechanisms of TCEP-induced hepatotoxicity are mostly unknown. We investigated the in vitro effects of TCEP as well as TCEP-induced cell growth in the L02 and HepG2 cells through the PI3K/Akt/mTOR pathway. We found that TCEP reduced cell viability of these cell lines, induced the cell growth arrest, upregulated mRNA and protein levels of SIRT1, and attenuated the PI3K/Akt/mTOR pathway. However, growth arrest of the L02 and HepG2 cells were aggravated after inhibiting the SIRT1 expression with EX-527. The findings above suggested that TCEP induced the cell growth arrest of L02 and HepG2 cells via attenuation of the SIRT1-independent PI3K/Akt/mTOR pathway. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26378621

  15. Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor ΚB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

    Directory of Open Access Journals (Sweden)

    Li J

    2014-03-01

    Full Text Available Jinchao Li,1,* Jun Li,2,* Ye Yue,1 Yiping Hu,1 Wenxiang Cheng,1 Ruoxi Liu,3 Xiaohua Pan,4 Peng Zhang1 1Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen, 2Emergency Surgery Department, Shaanxi Provincial People’s Hospital, Xi’an, 3Department of Orthopedics, Shandong University of Traditional Chinese Medicine, Jinan, 4Department of Orthopedics, Second Clinical Medical College, Jinan University, Shenzhen, People’ Republic of China *These authors contributed equally to this work Aims: Genistein, an isoflavone derivative found in soy, is known as a promising treatment for rheumatoid arthritis (RA. However, the detailed molecular mechanism of genistein in suppression of proinflammatory cytokine production remains ambiguous. The aim of this work was to evaluate the signal pathway by which genistein modulates inflammatory cytokine expression. Materials and methods: MH7A cells were stimulated with tumor necrosis factor (TNF-α and incubated with genistein, and interleukin (IL-1β, IL-6, and IL-8 production was measured by enzyme-linked immunosorbent assay. Nuclear translocation of nuclear factor (NF- ΚB was measured by a confocal fluorescence microscopy. The intracellular accumulation of reactive oxygen species (ROS was monitored using the fluorescent probe 5-6-chloromethyl-2´,7´-dichlorodihydrofluorescein diacetate. Signal-transduction protein expression was measured by Western blot. Results: Genistein decreased the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells in a dose-dependent manner. Genistein prevented TNF-α -induced NF-ΚB translocation as well as phosphorylation of IΚB kinase-α/β and IΚBα, and also suppressed TNF-α-induced AMPK inhibition. The production of IL-1β, IL-6, and IL-8 induced by TNF-α was decreased by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting that inhibition of Akt activation might

  16. Hypoxia induces phosphorylation of the cyclic AMP response element-binding protein by a novel signaling mechanism.

    Science.gov (United States)

    Beitner-Johnson, D; Millhorn, D E

    1998-07-31

    To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway. PMID:9677418

  17. Protective Effect of DHT on Apoptosis Induced by U18666A via PI3K/Akt Signaling Pathway in C6 Glial Cell Lines.

    Science.gov (United States)

    Yao, Kai; Wu, Junfeng; Zhang, Jianfeng; Bo, Jimei; Hong, Zhen; Zu, Hengbing

    2016-07-01

    Various useful animal models, such as Alzheimer's disease and Niemann-Pick disease, were provided by U18666A. However, the pathogenesis of U18666A-induced diseases, including U18666A-mediated apoptosis, remains incompletely elucidated, and therapeutic strategies are still limited. Dihydrotestosterone (DHT) has been reported to contribute to the prevention and treatment of neurodegenerative disorders. Our study investigated the neuroprotective activity of DHT in U18666A-related diseases. Apoptosis of C6 cells was detected by Hoechst 33258 fluorescent staining and flow cytometry with annexin V-FITC/PI dual staining. Cell viability was assessed using Cell Counting Kit-8. Expression of apoptosis-related proteins, such as Akt, seladin-1, Bcl-2 family proteins, and caspase-3, was determined using Western blot. Our results demonstrated that the apoptotic rate of C6 cells significantly increased after U18666A addition, but was remarkably reduced after DHT treatment. Pretreatment with DHT attenuated U18666A-induced cell viability loss. PI3K inhibitor LY294002 could suppress DHT anti-apoptotic effect. Furthermore, we discovered that U18666A could significantly downregulate seladin-1 expression in a dose-dependent manner, but no significant change was observed in Bcl-xL, Bax, and P-Akt protein expressions. Compared with U18666A-treated group, the expression of P-Akt, seladin-1, and Bcl-xL significantly increased, and the expression of Bax and caspase-3 remarkably reduced after DHT treatment. However, in the presence of LY294002, the effect of DHT was reversed. In conclusion, we found that seladin-1 may take part in U18666A-induced apoptosis. DHT may inhibit U18666A-induced apoptosis by regulating downstream apoptosis-related proteins including seladin-1, caspase-3, Bcl-xL, and Bax through activation of the PI3K/Akt signal pathway. PMID:26340949

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

    Science.gov (United States)

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

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

  19. Phosphatidylinositol 3-kinase/Akt pathway regulates hepatic stellate cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Xiao-Yu Jiang; Li Liu; Hui-Qing Jiang

    2008-01-01

    AIM:To investigate the role of phosphatidylinositol 3-kinase(PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells(HSC).METHODS:An activated HSC cell line was used in this study.LY 294002,the PI 3-K/Akt signal pathway blocker was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this pathway in HSC apoptosis.Immunocytochemistry,Western blot and reverse transcription polymerase chain reaction(RT-PCR)analysis were applied to detect the expression of PI 3-K,and simultaneously phosphorylated-Akt(p-Akt)and total-Akt were determined by Western blot.The HSC apoptosis was examined by annexin-V/propidium iodide double-labelled flow cytometry and transmission electron microscopy.RESULTS:The apoptosis rates in LY 294002(30.82% ±2.90%)and LY 294002+PDGF-BB(28.16%±2.58%)groups were significantly increased compared with those of control(9.02%±1.81%)and PDGF-BB(4.35%±1.18%).PDGF-BB augmented PI 3-K and p-Akt expression.LY 294002 significantly reduced the contents of PI 3-K and p-Akt.mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression.CONCLUSION:Inhibition of PI 3-K/Akt signaling pathway Induces apoptosis in HSC.(C)2008 The WJG Press.All rights reserved.

  20. The role of Akt on Arsenic trioxide suppression of 3T3-L1 preadipocyte differentiation

    Institute of Scientific and Technical Information of China (English)

    Zhi Xin WANG; Chun Sun JIANG; Lei LIU; Xiao Hui WANG; Hai Jing JIN; Qiao WU; Quan CHEN

    2005-01-01

    The present study investigates the molecular details of how arsenic trioxide inhibits preadipocyte differentiation and examines the role of Akt/PKB in regulation of differentiation and apoptosis. Continual exposure of arsenic trioxide, at the clinic achievable dosage that does not induce apoptosis, suppressed 3T3-L1 cell differentiation into fat cells by inhibiting the expression of PPARγ and C/EBPα and disrupting the interaction between PPARγ and RXRα, which determines the programming of the adipogenic genes. Interestingly, if we treated the cells for 12 or 24 h and then withdrew arsenic trioxide, the cells were able to differentiate to the comparable levels of untreated cells as assayed by the activity of GAPDH, the biochemical marker of preadipocyte differentiation. Long term treatment blocked the differentiation and the activity of GAPDH could not recover to the comparable levels of untreated cells. Continual exposure of arsenic trioxide caused accumulation in G2/M phase and the accumulation of p21. We found that arsenic trioxide induced the expression and the phosphorylation of Akt/PKB and it inhibited the interaction between Akt/PKB and PPARγ. Akt/PKB inhibitor appears to block the arsenic trioxide suppression of differentiation. Our results suggested that Akt/PKB may play a role in suppression of apoptosis and negatively regulate preadipocyte differentiation.

  1. Roles of Akt and SGK1 in the Regulation of Renal Tubular Transport

    Directory of Open Access Journals (Sweden)

    Nobuhiko Satoh

    2015-01-01

    Full Text Available A serine/threonine kinase Akt is a key mediator in various signaling pathways including regulation of renal tubular transport. In proximal tubules, Akt mediates insulin signaling via insulin receptor substrate 2 (IRS2 and stimulates sodium-bicarbonate cotransporter (NBCe1, resulting in increased sodium reabsorption. In insulin resistance, the IRS2 in kidney cortex is exceptionally preserved and may mediate the stimulatory effect of insulin on NBCe1 to cause hypertension in diabetes via sodium retention. Likewise, in distal convoluted tubules and cortical collecting ducts, insulin-induced Akt phosphorylation mediates several hormonal signals to enhance sodium-chloride cotransporter (NCC and epithelial sodium channel (ENaC activities, resulting in increased sodium reabsorption. Serum- and glucocorticoid-inducible kinase 1 (SGK1 mediates aldosterone signaling. Insulin can stimulate SGK1 to exert various effects on renal transporters. In renal cortical collecting ducts, SGK1 regulates the expression level of ENaC through inhibition of its degradation. In addition, SGK1 and Akt cooperatively regulate potassium secretion by renal outer medullary potassium channel (ROMK. Moreover, sodium-proton exchanger 3 (NHE3 in proximal tubules is possibly activated by SGK1. This review focuses on recent advances in understanding of the roles of Akt and SGK1 in the regulation of renal tubular transport.

  2. Conditional Activation of Akt in Adult Skeletal Muscle Induces Rapid Hypertrophy

    OpenAIRE

    Lai, Ka-Man V.; Gonzalez, Michael; Poueymirou, William T.; Kline, William O.; Na, Erqian; Zlotchenko, Elizabeth; Stitt, Trevor N.; Economides, Aris N.; Yancopoulos, George D.; Glass, David J.

    2004-01-01

    Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Ak...

  3. Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways.

    Science.gov (United States)

    Yan, Tingting; Zhao, Yan; Zhang, Xia; Lin, Xiaotong

    2016-03-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption. PMID:26978376

  4. Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Tingting Yan

    2016-03-01

    Full Text Available Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB. Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK and decrease of the level of activated extracellular signal-regulated kinases (ERKs. Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption.

  5. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    Science.gov (United States)

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  6. Nitric Oxide and Brassinosteroids Mediated Fungal Endophyte-Induced Volatile Oil Production Through Protein Phosphorylation Pathways in Atractylodes lancea Plantlets

    Institute of Scientific and Technical Information of China (English)

    Cheng-Gang Ren; Chuan-Chao Dai

    2013-01-01

    Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide (NO) and brassinolide (BL) in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. AL12 enhanced the activities of total protein phosphorylation, Ca2þ-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte-induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.

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

    Energy Technology Data Exchange (ETDEWEB)

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY (United States); Lee, Jeong-Chae [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY (United States); School of Dentistry and Institute of Oral Biosciences (BK21 program), Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Shi, Xianglin, E-mail: xshi5@email.uky.edu [Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY (United States)

    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

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

    International Nuclear Information System (INIS)

    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

  9. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Guo [Los Alamos National Laboratory; Mcmahon, Benjamin H [Los Alamos National Laboratory; Tung, Chang - Shung [Los Alamos National Laboratory

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  10. PI3K-AKT Signaling via Nrf2 Protects against Hyperoxia-Induced Acute Lung Injury, but Promotes Inflammation Post-Injury Independent of Nrf2 in Mice.

    Directory of Open Access Journals (Sweden)

    Narsa M Reddy

    Full Text Available Lung epithelial and endothelial cell death accompanied by inflammation contributes to hyperoxia-induced acute lung injury (ALI. Impaired resolution of ALI can promote and/or perpetuate lung pathogenesis, including fibrosis. Previously, we have shown that the transcription factor Nrf2 induces cytoprotective gene expression and confers protection against hyperoxic lung injury, and that Nrf2-mediated signaling is also crucial for the restoration of lung homeostasis post-injury. Although we have reported that PI3K/AKT signaling is required for Nrf2 activation in lung epithelial cells, significance of the PI3K/AKT-Nrf2 crosstalk during hyperoxic lung injury and repair remains unclear. Thus, we evaluated this aspect using Nrf2 knockout (Nrf2(-/- and wild-type (Nrf2(+/+ mouse models. Here, we show that pharmacologic inhibition of PI3K/AKT signaling increased lung inflammation and alveolar permeability in Nrf2(+/+ mice, accompanied by decreased expression of Nrf2-target genes such as Nqo1 and Hmox1. PI3K/AKT inhibition dampened hyperoxia-stimulated Nqo1 and Hmox1 expression in lung epithelial cells and alveolar macrophages. Contrasting with its protective effects, PI3K/AKT inhibition suppressed lung inflammation in Nrf2(+/+ mice during post-injury. In Nrf2(-/- mice exposed to room-air, PI3K/AKT inhibition caused lung injury and inflammation, but it did not exaggerate hyperoxia-induced ALI. During post-injury, PI3K/AKT inhibition did not augment, but rather attenuated, lung inflammation in Nrf2(-/- mice. These results suggest that PI3K/AKT-Nrf2 signaling is required to dampen hyperoxia-induced lung injury and inflammation. Paradoxically, the PI3K/AKT pathway promotes lung inflammation, independent of Nrf2, during post-injury.

  11. Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

    International Nuclear Information System (INIS)

    Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells

  12. IL-7 splicing variant IL-7{delta}5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Deshun [Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006 (China); Department of Pharmaceutical science, Guangdong Pharmaceutical University, Guangzhou, Guangdong (China); Liu, Bing [Department of Pharmaceutical science, Guangdong Pharmaceutical University, Guangzhou, Guangdong (China); Jin, Xiaobao [Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006 (China); Zhu, Jiayong, E-mail: zhujiayong888@163.com [Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006 (China)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer This study confirms the role of IL-7{delta}5 in breast cancer cell proliferation. Black-Right-Pointing-Pointer IL-7{delta}5 promotes breast cancer cell proliferation and cell cycle progression. Black-Right-Pointing-Pointer IL-7{delta}5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7{delta}5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7{delta}5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27{sup kip1} expression. Mechanistically, we found that IL-7{delta}5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7{delta}5. In conclusion, our findings demonstrate that IL-7{delta}5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7{delta}5 may be a potential target for human breast cancer therapeutics intervention.

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

    Science.gov (United States)

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

    2001-01-01

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

  14. Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

    International Nuclear Information System (INIS)

    In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase [A-kinase], from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from 32P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the 32P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase

  15. Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

    1987-05-01

    In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase (A-kinase), from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from /sup 32/P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the /sup 32/P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase.

  16. Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer.

    Science.gov (United States)

    Timsah, Z; Ahmed, Z; Ivan, C; Berrout, J; Gagea, M; Zhou, Y; Pena, G N A; Hu, X; Vallien, C; Kingsley, C V; Lu, Y; Hancock, J F; Liu, J; Gladden, A B; Mills, G B; Lopez-Berestein, G; Hung, M-C; Sood, A K; Bogdanov, M; Ladbury, J E

    2016-04-28

    In the absence of extracellular stimulation the adaptor protein growth factor receptor-bound protein (Grb2) and the phospholipase Plcγ1 compete for the same binding site on fibroblast growth factor receptor 2 (FGFR2). Reducing cellular Grb2 results in upregulation of Plcγ1 and depletion of the phospholipid PI(4,5)P2. The functional consequences of this event on signaling pathways are unknown. We show that the decrease in PI(4,5)P2 level under non-stimulated conditions inhibits PTEN activity leading to the aberrant activation of the oncoprotein Akt. This results in excessive cell proliferation and tumor progression in a xenograft mouse model. As well as defining a novel mechanism of Akt phosphorylation with important therapeutic consequences, we also demonstrate that differential expression levels of FGFR2, Plcγ1 and Grb2 correlate with patient survival. Oncogenesis through fluctuation in the expression levels of these proteins negates extracellular stimulation or mutation and defines them as novel prognostic markers in ovarian cancer. PMID:26212011

  17. Heat shock protein 27 regulates oxidative stress-induced apoptosis in cardiomyocytes:mechanisms via reactive oxygen species generation and Akt activation

    Institute of Scientific and Technical Information of China (English)

    LIU Li; ZHANG Xiao-jin; JIANG Su-rong; DING Zheng-nian; DING Guo-xian; HUANG Jun; CHENG Yun-lin

    2007-01-01

    Background Increased reactive oxygen species(ROS)formation,which in turn promotes cardiomyocytes apoptosis,is associated with the pathogenesis and progression of various cardiac diseases such as ischemia and heart failure.Recent studies have shown that over expression of heat shock protein 27(Hsp27)confers resistance to cardiac ischemia/reperfusion injury.However,not much is known about the regulation of myocyte survival by Hsp27.Methods The rat cardiac cell line H9c2,with a stable overexpression of Hsp27,was established,with empty vector transfected H9c2 cells as controls.Following the cells challenged by Hydrogen Peroxide(H2O2),lactate dehydrogenase (LDH)release,apoptosis,intracellular ROS,cell morphology,mitochondrial transmembrane potential and the activation of serine/threonine kinase Akt were determined.Results Along with marked suppression of H2O2-induced injury by Hsp27 overexpression in H9c2 cells,ROS generation and the loss of mitochondrial membrane potential were also significantly depressed.Furthermore,augmented Akt activation was observed in Hsp27 overexpressed H9c2 cells following H2O2 exposure.Conclusions Hsp27 inhibits oxidative stress-induced H9c2 damage and inhibition of ROS generation and the augmentation of Akt activation may be involved in the protective signaling.

  18. Akt is negatively regulated by the MULAN E3 ligase

    Institute of Scientific and Technical Information of China (English)

    Seunghee Bae; Jongdoo Kim; Hong-Duck Um; In-Chul Park; Su-Jae Lee; Seon Young Nam; Young-Woo Jin; Jae Ho Lee; Sungkwan An; Sun-Yong Kim; Jin Hyuk Jung; Yeongmin Yoon; Hwa Jun Cha; Hyunjin Lee; Karam Kim; Jongran Kim; In-Sook An

    2012-01-01

    The serine/threonine kinase Akt functions in multiple cellular processes,including cell survival and tumor development.Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation.In this study,we identified a negative regulator of Akt,MULAN,which possesses both a RING finger domain and E3 ubiquitin ligase activity.Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo.Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN.The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability.These data provide insight into the Akt ubiquitination signaling network.

  19. Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

    Directory of Open Access Journals (Sweden)

    Özel Cem

    2012-04-01

    Full Text Available Abstract Background The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-β/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. Results Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. Conclusions Here we show that hybrid cells could evolve exhibiting a

  20. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    International Nuclear Information System (INIS)

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, 32P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide

  1. Ginkgo Biloba Extract Ameliorates Oxidative Phosphorylation Performance and Rescues Aβ-Induced Failure

    Science.gov (United States)

    Rhein, Virginie; Giese, Maria; Baysang, Ginette; Meier, Fides; Rao, Stefania; Schulz, Kathrin L.; Hamburger, Matthias; Eckert, Anne

    2010-01-01

    Background Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer's disease (AD). Recently, we demonstrated that chronic exposure to amyloid-beta (Aβ) in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP) resulted in (i) activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS) and in (ii) a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE) is able to rescue Aβ-induced defects in energy metabolism. Methodology/Principal Findings We used a high-resolution respiratory protocol to evaluate OXPHOS respiratory capacity under physiological condition in control (stably transfected with the empty vector) and APP cells after treatment with GBE. In addition, oxygen consumption of isolated mitochondria, activities of mitochondrial respiratory enzymes, ATP and reactive oxygen species (ROS) levels as well as mitochondrial membrane mass and mitochondrial DNA content were determined. We observed a general antioxidant effect of GBE leading to an increase of the coupling state of mitochondria as well as energy homeostasis and a reduction of ROS levels in control cells and in APP cells. GBE effect on OXPHOS was even preserved in mitochondria after isolation from treated cells. Moreover, these functional data were paralleled by an up-regulation of mitochondrial DNA. Improvement of the OXPHOS efficiency was stronger in APP cells than in control cells. In APP cells, the GBE-induced amelioration of oxygen consumption most likely arose from the modulation and respective normalization of the Aβ-induced disturbance in the activity of mitochondrial complexes III and IV restoring impaired ATP levels possibly through decreasing Aβ and oxidative stress level. Conclusions/Significance Although the underlying

  2. Ginkgo biloba extract ameliorates oxidative phosphorylation performance and rescues abeta-induced failure.

    Directory of Open Access Journals (Sweden)

    Virginie Rhein

    Full Text Available BACKGROUND: Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer's disease (AD. Recently, we demonstrated that chronic exposure to amyloid-beta (Abeta in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP resulted in (i activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS and in (ii a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE is able to rescue Abeta-induced defects in energy metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We used a high-resolution respiratory protocol to evaluate OXPHOS respiratory capacity under physiological condition in control (stably transfected with the empty vector and APP cells after treatment with GBE. In addition, oxygen consumption of isolated mitochondria, activities of mitochondrial respiratory enzymes, ATP and reactive oxygen species (ROS levels as well as mitochondrial membrane mass and mitochondrial DNA content were determined. We observed a general antioxidant effect of GBE leading to an increase of the coupling state of mitochondria as well as energy homeostasis and a reduction of ROS levels in control cells and in APP cells. GBE effect on OXPHOS was even preserved in mitochondria after isolation from treated cells. Moreover, these functional data were paralleled by an up-regulation of mitochondrial DNA. Improvement of the OXPHOS efficiency was stronger in APP cells than in control cells. In APP cells, the GBE-induced amelioration of oxygen consumption most likely arose from the modulation and respective normalization of the Abeta-induced disturbance in the activity of mitochondrial complexes III and IV restoring impaired ATP levels possibly through decreasing Abeta and oxidative stress level. CONCLUSIONS

  3. Quantitative Analysis of Cytokine-Induced Hepatocyte Nuclear Factor-4α Phosphorylation by Mass Spectrometry

    OpenAIRE

    Wang, Zhongyan; Salih, Erdjan; Burke, Peter A.

    2011-01-01

    Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored. To address these issues, we have carried out an extensive quantitative ...

  4. SchA-p85-FAK complex dictates isoform-specific activation of Akt2 and subsequent PCBP1-mediated post-transcriptional regulation of TGFβ-mediated epithelial to mesenchymal transition in human lung cancer cell line A549.

    Science.gov (United States)

    Xue, Xinying; Wang, Xin; Liu, Yuxia; Teng, Guigen; Wang, Yong; Zang, Xuefeng; Wang, Kaifei; Zhang, Jinghui; Xu, Yali; Wang, Jianxin; Pan, Lei

    2014-08-01

    A post-transcriptional pathway by which TGF-β modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-β for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-β induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-β treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-β-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma. PMID:24819169

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

    Directory of Open Access Journals (Sweden)

    Huawang Sun

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

  6. PI-103 and Quercetin Attenuate PI3K-AKT Signaling Pathway in T- Cell Lymphoma Exposed to Hydrogen Peroxide

    Science.gov (United States)

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2016-01-01

    Phosphatidylinositol 3 kinase—protein kinase B (PI3K-AKT) pathway has been considered as major drug target site due to its frequent activation in cancer. AKT regulates the activity of various targets to promote tumorigenesis and metastasis. Accumulation of reactive oxygen species (ROS) has been linked to oxidative stress and regulation of signaling pathways for metabolic adaptation of tumor microenvironment. Hydrogen peroxide (H2O2) in this context is used as ROS source for oxidative stress preconditioning. Antioxidants are commonly considered to be beneficial to reduce detrimental effects of ROS and are recommended as dietary supplements. Quercetin, a ubiquitous bioactive flavonoid is a dietary component which has attracted much of interest due to its potential health-promoting effects. Present study is aimed to analyze PI3K-AKT signaling pathway in H2O2 exposed Dalton’s lymphoma ascite (DLA) cells. Further, regulation of PI3K-AKT pathway by quercetin as well as PI-103, an inhibitor of PI3K was analyzed. Exposure of H2O2 (1mM H2O2 for 30min) to DLA cells caused ROS accumulation and resulted in increased phosphorylation of PI3K and downstream proteins PDK1 and AKT (Ser-473 and Thr-308), cell survival factors BAD and ERK1/2, as well as TNFR1. However, level of tumor suppressor PTEN was declined. Both PI-103 & quercetin suppressed the enhanced level of ROS and significantly down-regulated phosphorylation of AKT, PDK1, BAD and level of TNFR1 as well as increased the level of PTEN in H2O2 induced lymphoma cells. The overall result suggests that quercetin and PI3K inhibitor PI-103 attenuate PI3K-AKT pathway in a similar mechanism. PMID:27494022

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

    Energy Technology Data Exchange (ETDEWEB)

    Horiuchi, Rie [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Hong, Zhang [Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Ushida, Takashi [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan)

    2012-08-15

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

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

    International Nuclear Information System (INIS)

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

  9. Astaxanthin Alleviates Early Brain Injury Following Subarachnoid Hemorrhage in Rats: Possible Involvement of Akt/Bad Signaling

    Directory of Open Access Journals (Sweden)

    Xiang-Sheng Zhang

    2014-07-01

    Full Text Available Apoptosis has been proven to play a crucial role in early brain injury pathogenesis and to represent a target for the treatment of subarachnoid hemorrhage (SAH. Previously, we demonstrated that astaxanthin (ATX administration markedly reduced neuronal apoptosis in the early period after SAH. However, the underlying molecular mechanisms remain obscure. In the present study, we tried to investigate whether ATX administration is associated with the phosphatidylinositol 3-kinase-Akt (PI3K/Akt pathway, which can play an important role in the signaling of apoptosis. Our results showed that post-SAH treatment with ATX could cause a significant increase of phosphorylated Akt and Bad levels, along with a significant decrease of cleaved caspase-3 levels in the cortex after SAH. In addition to the reduced neuronal apoptosis, treatment with ATX could also significantly reduce secondary brain injury characterized by neurological dysfunction, cerebral edema and blood-brain barrier disruption. In contrast, the PI3K/Akt inhibitor, LY294002, could partially reverse the neuroprotection of ATX in the early period after SAH by downregulating ATX-induced activation of Akt/Bad and upregulating cleaved caspase-3 levels. These results provided the evidence that ATX could attenuate apoptosis in a rat SAH model, potentially, in part, through modulating the Akt/Bad pathway.

  10. Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS

    Science.gov (United States)

    Sun, Li-na; Liu, Xiang-chun; Chen, Xiang-jun; Guan, Guang-ju; Liu, Gang

    2016-01-01

    Aim: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. Methods: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. Results: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 μmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. Conclusion: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS. PMID:26838071

  11. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

    Czech Academy of Sciences Publication Activity Database

    Li, P.; Hulák, M.; Li, Z.; H.; Šulc, Miroslav; Pšenička, M.; Rodina, M.; Gela, D.; Linhart, O.

    2013-01-01

    Roč. 80, č. 2 (2013), s. 84-89. ISSN 0093-691X Institutional support: RVO:61388971 Keywords : Cryopreservation * Sperm * Phosphorylation Subject RIV: CE - Biochemistry Impact factor: 1.845, year: 2013

  12. Downregulation of IL-6-induced STAT3 tyrosine phosphorylation by TGF-β1 is mediated by caspase-dependent and -independent processes

    NARCIS (Netherlands)

    Wierenga, A.T.J.; Schuringa, J.J.; Eggen, B.J.L.; Kruijer, W.; Vellenga, E.

    2002-01-01

    To explore the possible cross-talk between the IL-6 and TGF-β1 pathways in AML blast cells, the effect of TGF-β1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-β1 pretreatment was observed in four out of 40 AML cases (

  13. Downregulation of IL-6-induced STAT3 tyrosine phosphorylation by TGF-beta 1 is mediated by caspase-dependent and -independent processes

    NARCIS (Netherlands)

    Wierenga, ATJ; Schuringa, JJ; Eggen, BJL; Kruijer, W; Vellenga, E

    2002-01-01

    To explore the possible cross-talk between the IL-6 and TGF-beta1 pathways in AML blast cells, the effect of TGF-beta1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-beta1 pretreatment was observed in four out of 40 AM

  14. Relationship between G-proteins associated transmembrane cascades and MAPK phosphorylation induced by recombinant interleukin-α in fibroblast-like synoviocytes from rats with adjuvant arthritis

    Institute of Scientific and Technical Information of China (English)

    Yong-qiuZHENG; WeiWEI; LeiZHU; Xiao-yiJIA

    2004-01-01

    AIM: To elucidate the relationship between G protein-associ-ated transmembrane cascade and mitogen-activated protein kinases (MAPKs) phosphorylation in recombinant rat IL-1α(rIL-1α)-induced fibroblast-like synoviocytes (FLS) from rats with adjuvant arthritis (AA). METHODS: The expressions of MAPKs phosphorylation, the stimulatory subunit of G alpha

  15. A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Xiaoxue Qiu

    2012-06-01

    Full Text Available Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3 are inhibitors of the Janus tyrosine kinase (JAK/signal transducers and activators of transcription (STAT pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-XL protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

  16. Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

    Science.gov (United States)

    Cadete, Virgilio J. J.; Sawicka, Jolanta; Jaswal, Jagdip; Lopaschuk, Gary D.; Schulz, Richard; Szczesna-Cordary, Danuta; Sawicki, Grzegorz

    2012-01-01

    Summary Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. PMID:22564771

  17. Piperlongumine induces apoptosis and autophagy in human lung cancer cells through inhibition of PI3K/Akt/mTOR pathway.

    Science.gov (United States)

    Wang, Feng; Mao, Yong; You, Qingjun; Hua, Dong; Cai, Dongyan

    2015-09-01

    Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anti-cancer effects. Nonetheless, the anti-tumor effect of PL in lung cancer cells still remains unclear. In the present study, we reported the chemotherapeutic effects of PL using in vitro and in vivo models. We showed that PL displayed potent anti-neoplastic activity against lung cancer A549 cells as well as corresponding docetaxel-resistant A549/DTX cells. In addition, we found that PL induced apoptosis in both A549 and A549/DTX cells. PL also induced autophagy in A549/DTX cells. Moreover, autophagy-specific inhibitors (3-methyladenine) or Beclin1 and Atg 5 small interfering RNAs (siRNAs) enhanced PL-induced apoptosis, indicating that PL-mediated autophagy may protect A549/DTX cells from undergoing apoptotic cell death. Furthermore, we observed the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway by PL. Finally, PL inhibited the growth of A549/DTX xenograft tumors, which was associated with inhibition of cell proliferation, induction of apoptosis of tumor cells and decreased expression of p-Akt and p-mTOR in tumor xenograft tissues. In summary, our study demonstrated that PL induced apoptosis and autophagy through modulation of the PI3K/Akt/mTOR pathway in human lung cancer cells. This study may provide a rationale for future clinical application using PL as a chemotherapeutic agent for lung cancer. PMID:26246196

  18. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Science.gov (United States)

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage. PMID:26465605

  19. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Sam Coffey

    Full Text Available Diabetes mellitus (DM has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the