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Sample records for 3-kinase inhibitor ly294002

  1. Synergistic inhibition of colon carcinoma cell growth by Hedgehog-Gli1 inhibitor arsenic trioxide and phosphoinositide 3-kinase inhibitor LY294002.

    PubMed

    Cai, Xinyi; Yu, Kun; Zhang, Lijuan; Li, Yunfeng; Li, Qiang; Yang, Zhibin; Shen, Tao; Duan, Lincan; Xiong, Wei; Wang, Weiya

    2015-01-01

    The Hedgehog (Hh) signaling pathway not only plays important roles in embryogenesis and adult tissue homeostasis, but also in tumorigenesis. Aberrant Hh pathway activation has been reported in a variety of malignant tumors including colon carcinoma. Here, we sought to investigate the regulation of the Hh pathway transcription factor Gli1 by arsenic trioxide and phosphoinositide 3-kinase (PI3K) inhibitor LY294002 in colon carcinoma cells. We transfected cells with siGli1 and observed a significant reduction of Gli1 expression in HCT116 and HT29 cells, which was confirmed by quantitative real-time polymerase chain reaction and Western blots. Knocking down endogenous Gli1 reduced colon carcinoma cell viability through inducing cell apoptosis. Similarly, knocking down Gli2 using short interfering RNA impaired colon carcinoma cell growth in vitro. To elucidate the regulation of Gli1 expression, we found that both Gli inhibitor arsenic trioxide and PI3K inhibitor LY294002 significantly reduced Gli1 protein expression and colon carcinoma cell proliferation. Arsenic trioxide treatment also reduced Gli1 downstream target gene expression, such as Bcl2 and CCND1. More importantly, the inhibition of Hedgehog-Gli1 by arsenic trioxide showed synergistic anticancer effect with the PI3K inhibitor LY294002 in colon carcinoma cells. Our findings suggest that the Hh pathway transcription factor Gli1 is involved in the regulation of colon carcinoma cell viability. Inhibition of Hedgehog-Gli1 expression by arsenic trioxide and PI3K inhibitor synergistically reduces colon cancer cell proliferation, indicating that they could be used as an effective anti-colon cancer combination therapy.

  2. PI3K inhibitors LY294002 and IC87114 reduce inflammation in carrageenan-induced paw oedema and down-regulate inflammatory gene expression in activated macrophages.

    PubMed

    Eräsalo, Heikki; Laavola, Mirka; Hämäläinen, Mari; Leppänen, Tiina; Nieminen, Riina; Moilanen, Eeva

    2015-01-01

    PI3K/Akt pathway is a well-characterized pathway controlling cellular processes such as proliferation, migration and survival, and its role in cancer is vastly studied. There is also evidence to suggest the involvement of this pathway in the regulation of inflammatory responses. In this study, an attempt was made to investigate the role of PI3Ks in acute inflammation in vivo using pharmacological inhibitors against PI3Ks in the carrageenan-induced paw oedema model. A non-selective PI3K inhibitor LY294002 and a PI3Kδ-selective inhibitor IC87114 were used. Both of these inhibitors reduced inflammatory oedema upon carrageenan challenge in the mouse paw. To explain this result, the effects of the two inhibitors on inflammatory gene expression were investigated in activated macrophages. LY294002 and IC87114 prevented Akt phosphorylation as expected and down-regulated the expression of inflammatory factors IL-6, MCP-1,TNFα and iNOS. These findings suggest that PI3K inhibitors could be used to attenuate inflammatory responses and that the mechanism of action behind this effect is the down-regulation of inflammatory gene expression.

  3. Differential modulation of mitogen driven proliferation and homeostasis driven proliferation of T cells by rapamycin, Ly294002 and chlorophyllin.

    PubMed

    Sharma, Deepak; Kumar, Sandur Santosh; Raghu, Rashmi; Khanam, Shazia; Sainis, Krishna Balaji

    2007-04-01

    Homeostasis driven proliferation (HDP) of naïve CD4+ T cells depends upon T cell receptor ligation with self-MHC II along with availability of interleukin-7. But the exact nature of downstream signaling events involved in HDP of helper T cells remains elusive. To identify the specific involvement of signaling molecules in HDP, purified CD4+ T cells were treated with either mTOR inhibitor rapamycin or PI3kinase inhibitor Ly294002 or with an antioxidant chlorophyllin (CHL) in vitro. Rapamycin treated cells failed to proliferate, expressed anergic T cell specific transcription factor genes egr-2 and egr-3 and showed diminished IFN-gamma production in response to Con A stimulation in vitro. Although CHL treated cells also failed to proliferate, they showed a normal IFN-gamma production during primary stimulation and did not upregulate egr-2 and egr-3 genes following restimulation in vitro. Ly294002 treated cells failed to express IL-2 and IFN-gamma and did not divide in response to Con A stimulation in vitro. While all these inhibitors significantly inhibited CD4+ T cell proliferation in response to the mitogen in vitro, only CHL treatment could inhibit their HDP in lymphopenic mice. Our results also demonstrate that combined treatment with rapamycin and Ly294002 did not inhibit HDP of CD4+ T cells. Thus, the present study, for the first time, shows a non-essential role of mTOR and PI3kinase during HDP of CD4+ T cells and also describes its possible regulation by an antioxidant.

  4. Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002.

    PubMed

    Jacobs, Marc D; Black, James; Futer, Olga; Swenson, Lora; Hare, Brian; Fleming, Mark; Saxena, Kumkum

    2005-04-08

    Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.

  5. LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells.

    PubMed

    Pistilli, Alessandra; Rende, Mario; Crispoltoni, Lucia; Montagnoli, Claudia; Stabile, Anna Maria

    2015-01-01

    Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without β-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

  6. Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death

    PubMed Central

    Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.

    2013-01-01

    Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after

  7. ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor identified using the JFCR39 drug discovery system.

    PubMed

    Kong, De-xin; Yamori, Takao

    2010-09-01

    JFCR39 is an informatic anticancer drug discovery system that utilizes a panel of 39 human cancer cells coupled with a drug-activity database. This system not only provides disease-oriented information but can also predict the mechanism of action of a given antitumor agent. Development of a phosphatidylinositol 3-kinase (PI3K) inhibitor as an anticancer drug candidate has attracted a great deal of attention from both academia and industry because PI3K is known to be closely involved in carcinogenesis. ZSTK474 was identified as a PI3K inhibitor using JFCR39 system in combination with COMPARE analysis program. These findings were based on the similar fingerprint (growth inhibition profiles for JFCR39 human cancer cell line panel) with that of a classical PI3K inhibitor LY294002. Biochemical experiments confirmed ZSTK474 to be a potent pan-class I PI3K inhibitor, with high selectivity over other classes of PI3K and protein kinases. We previously reported the in vitro and in vivo antitumor efficacy of ZSTK474, together with the G(0)/G(1) arrest and antiangiogenic activity. Here, we review the JFCR39 system and summarize recent studies on PI3K biology and the development of PI3K inhibitors before discussing ZSTK474 in some detail.

  8. IPD-196, a novel phosphatidylinositol 3-kinase inhibitor with potent anticancer activity against hepatocellular carcinoma.

    PubMed

    Lee, Ju-Hee; Lee, Hyunseung; Yun, Sun-Mi; Jung, Kyung Hee; Jeong, Yujeong; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

    2013-02-01

    As the activation of phosphatidylinositol 3-kinase (PI3K) is associated with a wide variety of human malignancies, it is emerging as an attractive target for cancer treatment. In this study we synthesized a novel PI3Kα inhibitor, IPD-196 [ethyl 6-(5-(2,4-difluorophenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate], and evaluated its anticancer effects on human hepatocellular carcinoma (HCC) cells. IPD-196 effectively inhibited the phosphorylation of downstream PI3K effectors such as Akt, mTOR, p70S6K, and 4E-BP1, and its antiproliferative effect was more potent than that of sorafenib or LY294002. It also induced cell cycle arrest at the G0/G1 phase as well as apoptosis by increasing the proportion of sub-G1 apoptotic cells, and the levels of cleaved PARP, caspase-3, and caspase-9. Furthermore, it decreased the expression of HIF-1α and VEGF in Huh-7 cells, and inhibited tube formation and migration of human umbilical vein endothelial cells, which was confirmed by a Matrigel plug assay in mice. Taken together, IPD-196 exhibited its anticancer activity through disruption of the PI3K/Akt pathway that caused cell cycle arrest, apoptosis induction, and inhibition of angiogenesis in human HCC cells. We therefore suggest that IPD-196 may be a potential candidate drug for targeted HCC therapy.

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

    PubMed

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

    2016-11-01

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

  10. Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

    SciTech Connect

    Neuhof, Dirk Zwicker, Felix; Kuepper, Jan-Heiner; Debus, Juergen; Weber, Klaus-Josef

    2007-11-01

    Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.

  11. Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

    SciTech Connect

    McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H.

    2010-11-15

    Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G{sub 1} phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}; and knockdown of p27{sup kip1} with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

  12. Low [Mg2+]e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension.

    PubMed

    Northcott, Carrie A; Watts, Stephanie W

    2004-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been implicated in low extracellular Mg2+ concentration ( [Mg2+]e)-induced aortic contraction, and Mg2+ deficiency has been associated with hypertension. Moreover, arterial PI3K activity is increased in hypertensive deoxycorticosterone (DOCA)-salt rats. We hypothesized that low [Mg2+]e activates PI3K, eliciting enhanced vascular contraction, PI3K activity, and norepinephrine (NE)-induced contraction. Spontaneous tone was monitored in endothelium-denuded aortic strips from sham and DOCA-salt rats exposed to low Mg2+ (0.15 mmol/L), high Mg2+ (4.8 mmol/L), or normal (1.17 mmol/L) physiologic salt solution (PSS) in isolated tissue baths. LY294002 (20 micromol/L), a PI3K inhibitor, or vehicle was added (30 minutes), followed by NE (10(-9) to 3 x10(-5) mol/L). Low [Mg2+]e significantly enhanced tone in aortas from DOCA-salt and sham rats compared with normal PSS (DOCA-salt low [Mg2+]e, +51.5 +7.0 vs DOCA-salt normal PSS, +7.1 +1.4 % of initial phenylephrine [PE] contraction). LY294002 and incubation with high Mg2+ PSS decreased tone in aortas from DOCA-salt rats (low [Mg2+]e LY294002, --87.5 +8.8; normal PSS LY294002, -81.7 +13.7; and high [Mg2+]e, -31.2 +10.8 % of initial PE contraction). Low [Mg2+]e leftward-shifted NE-induced aortic contractions in sham and thus matched the shift observed with DOCA (-log EC50 mol/L: sham PSS, -7.7 +0.1; DOCA-salt PSS, -8.2 +0.1; sham low [Mg2+]e, -8.2 +0.1; and DOCA-salt low [Mg2+]e, -8.1 +0.1). Moreover, this shift was inhibited by LY294002. In conclusion, low [Mg2+]e might activate PI3K, leading to enhanced tone and agonist-induced contraction observed in aortas from DOCA-salt hypertensive rats.

  13. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  14. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  15. Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment

    PubMed Central

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds. PMID:23232172

  16. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  17. Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics

    PubMed Central

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate diverse cellular processes including proliferation, adhesion, survival, and motility. Dysregulated PI3K pathway signaling occurs in one-third of human tumors. Aberrantly activated PI3K signaling also confers sensitivity and resistance to conventional therapies. PI3K has been recognized as an attractive molecular target for novel anti-cancer molecules. In the last few years, several classes of potent and selective small molecule PI3K inhibitors have been developed, and at least fifteen compounds have progressed into clinical trials as new anticancer drugs. Among these, idelalisib has advanced to phase III trials in patients with advanced indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. In this review, we summarized the major molecules of PI3K signaling pathway, and discussed the preclinical models and clinical trials of potent small-molecule PI3K inhibitors. PMID:24261963

  18. Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

    PubMed Central

    Juvekar, Ashish; Hu, Hai; Yadegarynia, Sina; Lyssiotis, Costas A.; Ullas, Soumya; Lien, Evan C.; Bellinger, Gary; Son, Jaekyoung; Hok, Rosanna C.; Seth, Pankaj; Daly, Michele B.; Kim, Baek; Scully, Ralph; Asara, John M.; Cantley, Lewis C.; Wulf, Gerburg M.

    2016-01-01

    We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors. PMID:27402769

  19. Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

    PubMed

    Numakawa, Yumiko; Numakawa, Tadahiro; Matsumoto, Tomoya; Yagasaki, Yuki; Kumamaru, Emi; Kunugi, Hiroshi; Taguchi, Takahisa; Niki, Etsuo

    2006-05-01

    The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.

  20. Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

    PubMed

    Kubota, Y; Tanaka, T; Ohnishi, H; Kitanaka, A; Okutani, Y; Taminato, T; Ishida, T; Kamano, H

    2004-06-01

    In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

  1. The phosphoinositide 3-kinase signaling pathway is involved in the control of modified low-density lipoprotein uptake by human macrophages.

    PubMed

    Michael, Daryn R; Davies, Thomas S; Laubertová, Lucia; Gallagher, Hayley; Ramji, Dipak P

    2015-03-01

    The transformation of macrophages into lipid-loaded foam cells is a critical early event in the pathogenesis of atherosclerosis. Both receptor-mediated uptake of modified LDL, mediated primarily by scavenger receptors-A (SR-A) and CD36 along with other proteins such as lipoprotein lipase (LPL), and macropinocytosis contribute to macrophage foam cell formation. The signaling pathways that are involved in the control of foam cell formation are not fully understood. In this study, we have investigated the role of phosphoinositide 3-kinase (PI3K) in relation to foam cell formation in human macrophages. The pan PI3K inhibitor LY294002 attenuated the uptake of modified LDL and macropinocytosis, as measured by Lucifer Yellow uptake, by human macrophages. In addition, the expression of SR-A, CD36 and LPL was attenuated by LY294002. The use of isoform-selective PI3K inhibitors showed that PI3K-β, -γ and -δ were all required for the expression of SR-A and CD36 whereas only PI3K-γ was necessary in the case of LPL. These studies reveal a pivotal role of PI3K in the control of macrophage foam cell formation and provide further evidence for their potential as therapeutic target against atherosclerosis.

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

    PubMed Central

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

    1997-01-01

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

  3. Rapamycin regulates connective tissue growth factor expression of lung epithelial cells via phosphoinositide 3-kinase.

    PubMed

    Xu, Xuefeng; Wan, Xuan; Geng, Jing; Li, Fei; Yang, Ting; Dai, Huaping

    2013-09-01

    The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains largely unknown. It is believed that IPF is mainly driven by activated alveolar epithelial cells that have a compromised migration capacity, and that also produce substances (such as connective tissue growth factor, CTGF) that contribute to fibroblast activation and matrix protein accumulation. Because the mechanisms regulating these processes are unclear, the aim of this study was to determine the role of rapamycin in regulating epithelial cell migration and CTGF expression. Transformed epithelial cell line A549 and normal human pulmonary alveolar or bronchial epithelial cells were cultured in regular medium or medium containing rapamycin. Real time reverse transcriptase polymerase chain reaction was employed to determine CTGF mRNA expression. Western blotting and an enzyme-linked immunosorbent assay were used for detecting CTGF protein. Wound healing and migration assays were used to determine the cell migration potential. Transforming growth factor (TGF)-β type I receptor (TβRI) inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. It was found that treatment of A549 and normal human alveolar or bronchial epithelial cells with rapamycin significantly promoted basal or TGF-β1 induced CTGF expression. LY294002, not SB431542 attenuated the promotional effect of rapamycin on CTGF expression. Cell mobility was not affected by rapamycin in wound healing and migration assays. These data suggest rapamycin has a profibrotic effect in vitro and underscore the potential of combined therapeutic approach with PI3K and mammalian target of rapamycin inhibitors for the treatment of animal or human lung fibrosis.

  4. Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma.

    PubMed

    Grugan, Katharine D; Ma, Chunguang; Singhal, Seema; Krett, Nancy L; Rosen, Steven T

    2008-06-01

    Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

  5. PHOSPHOINOSITIDE 3-KINASE REGULATES THE ROLE OF RETROMER IN TRANSCYTOSIS OF THE POLYMERIC IMMUNOGLOBULIN RECEPTOR

    PubMed Central

    Vergés, Marcel; Sebastián, Isabel; Mostov, Keith E.

    2007-01-01

    Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer’s role in pIgR-pIgA transcytosis. PMID:17184770

  6. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    PubMed

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  7. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    PubMed Central

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2015-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  8. Phosphatidylinositol 3-kinase, MEK-1 and p38 mediate leptin/interferon-gamma synergistic NOS type II induction in chondrocytes.

    PubMed

    Otero, Miguel; Lago, Rocío; Gómez, Rodolfo; Lago, Francisca; Gomez-Reino, Juan Jesús; Gualillo, Oreste

    2007-10-27

    In a previous study, we established that leptin acts synergistically with interferon-gamma in inducing nitric oxide synthase type II in cultured chondrocytes via Janus kinase-2 activation. However, the exact molecular mechanism that accounts for this synergism is not completely understood. The aim of the present study was to further delineate the signalling pathway used by leptin/interferon-gamma in the nitric oxide synthase type II induction in chondrocytes. Consequently, the roles of PI-3 kinase, MEK1 and p38 kinase were investigated using specific pharmacological inhibitors (Wortmannin, LY 294002, PD 098,059 and SB 203580). For this purpose, the amount of stable nitrite, the end product of NO generation by activated chondrocytes, has been evaluated by Griess colorimetric reaction in culture medium of human primary chondrocytes and in the murine ATDC5 cell line stimulated with leptin (400 nM) and interferon-gamma (1 ng/ml), alone or in combination. Specific inhibitors for PI-3K, MEK1 and p38 were added 1 h before stimulation. Nitric oxide synthase type II mRNA was investigated by real-time RT-PCR and NOS type II protein expression has been evaluated by western blot analysis. Our results showed that, as expected, leptin synergizes with IFN-gamma in inducing NO accumulation in the supernatant of co-stimulated cells. Pre-treatment with Wortmannin, LY 294002, PD 098,059 and SB 203580 caused a significant decrease in nitrite production, NOS type II protein expression and NOS type II mRNA expression induced by leptin and interferon-gamma co-stimulation. These findings were confirmed in 15 and 21-day differentiated ATDC5 cells, and in normal human primary chondrocytes. This is the first report showing that NOS type II induction triggered by co-stimulation with leptin and interferon-gamma is mediated by a signaling pathway involving PI-3K, MEK1 and p38.

  9. Recent development of ATP-competitive small molecule phosphatidylinostitol-3-kinase inhibitors as anticancer agents

    PubMed Central

    Liu, Yu; Wan, Wen-zhu; Li, Yan; Zhou, Guan-lian; Liu, Xin-guang

    2017-01-01

    Phosphatidylinostitol-3-kinase (PI3K) is the potential anticancer target in the PI3K/Akt/ mTOR pathway. Here we reviewed the ATP-competitive small molecule PI3K inhibitors in the past few years, including the pan Class I PI3K inhibitors, the isoform-specific PI3K inhibitors and/or the PI3K/mTOR dual inhibitors. PMID:27769061

  10. Clinical development of phosphatidylinositol-3 kinase pathway inhibitors.

    PubMed

    Arteaga, Carlos L

    2010-01-01

    The PI3K pathway is the most commonly altered in human cancer. Several recent phase I studies with therapeutic inhibitors of this pathway have shown that pharmacological inhibition of PI3K in humans is feasible and overall well tolerated. Furthermore, there has already been clinical evidence of anti-tumor activity in patients with advanced cancer. The intensity and duration of PI3K inhibition required for an antitumor effect and the optimal pharmacodynamic biomarker(s) of pathway inactivation remain to be established. Preclinical and early clinical data support focusing on trials with PI3K inhibitors that are at a minimum enriched with patients with alterations in this signaling pathway. These inhibitors are likely to be more effective in combination with established and other novel molecular therapies.

  11. Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway.

    PubMed

    Yang, Heping; Wu, Dapeng; Zhang, Xiaojie; Wang, Xiang; Peng, Yi; Hu, Zhiping

    2012-10-05

    Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

  12. Neuroprotective Role of the PI3 Kinase/Akt Signaling Pathway in Zebrafish

    PubMed Central

    Chen, Shuang; Liu, Yunzhang; Rong, Xiaozhi; Li, Yun; Zhou, Jianfeng; Lu, Ling

    2017-01-01

    Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin-like growth factor-1), the ability to regulate cell survival has been attributed to the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of myristoylated Akt (myr-Akt), a constitutive form of Akt. Further analysis reveals that expressing exogenous myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis. PMID:28228749

  13. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy.

    PubMed

    Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

    2011-11-11

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors.

  14. Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3-kinase γ.

    PubMed

    Collier, Philip N; Martinez-Botella, Gabriel; Cornebise, Mark; Cottrell, Kevin M; Doran, John D; Griffith, James P; Mahajan, Sudipta; Maltais, François; Moody, Cameron S; Huck, Emilie Porter; Wang, Tiansheng; Aronov, Alex M

    2015-01-08

    Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.

  15. Hepatocyte growth factor (HGF) enhances cardiac commitment of differentiating embryonic stem cells by activating PI3 kinase

    SciTech Connect

    Roggia, Cristiana; Ukena, Christian; Boehm, Michael; Kilter, Heiko . E-mail: kilter@med-in.uni-saarland.de

    2007-03-10

    Hepatocyte growth factor (HGF) is a pleiotropic cytokine promoting proliferation, migration and survival in several cell types. HGF and its cognate receptor c-Met are expressed in cardiac cells during early cardiogenesis, but data concerning its role in cardiac differentiation of embryonic stem cells (ESCs) and the underlying molecular mechanisms involved are limited. In the present study we show that HGF significantly increases the number of beating embryoid bodies of differentiating ESCs without affecting beating frequency. Furthermore, HGF up-regulates the expression of the cardiac-specific transcription factors Nkx 2.5 and GATA-4 and of markers of differentiated cardiomyocytes, i.e. {alpha}-MHC, {beta}-MHC, ANF, MLC2v and Troponin T. The HGF-induced increase in Nkx 2.5 expression was inhibited by co-treatment with the PI3 kinase inhibitors Wortmannin and LY294002, but not by its inactive homolog LY303511, suggesting an involvement of the PI3 kinase/Akt pathway in this effect. We conclude that HGF is an important growth factor involved in cardiac differentiation and/or proliferation of ESCs and may therefore be critical for the in vitro generation of pre- or fully differentiated cardiomyocytes as required for clinical use of embryonic stem cells in cardiac diseases.

  16. Roles of tyrosine kinase-, 1-phosphatidylinositol 3-kinase-, and mitogen-activated protein kinase-signaling pathways in ethanol-induced contractions of rat aortic smooth muscle: possible relation to alcohol-induced hypertension.

    PubMed

    Yang, Zhi-wei; Wang, Jun; Zheng, Tao; Altura, Bella T; Altura, Burton M

    2002-08-01

    Insights into the relations between and among ethanol-induced contractions in rat aorta, tyrosine kinases (including src family of cytoplasmic tyrosine kinases), 1-phosphatidylinositol 3-kinases (PI-3Ks), mitogen-activated protein kinases (MAPKs), and regulation of intracellular free Ca(2+) ([Ca(2+)](i)) were investigated in the present study. Ethanol-induced concentration-dependent contractions in isolated rat aortic rings were attenuated greatly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein), an src homology domain 2 (SH2) inhibitor peptide, a highly specific antagonist of p38 MAPK (SB-203580), a potent, selective antagonist of two specific MAPK kinases-MEK1/MEK2 (U0126)-and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059), as well as by treatment with wortmannin or LY-294002 (both are selective antagonists of PI-3Ks). Inhibitory concentration 50 (IC(50)) levels obtained for these seven antagonists were consistent with reported inhibition constant (Ki) values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol-induced transient and sustained increases in [Ca(2+)](i) in primary single smooth muscle cells from rat aorta were markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, PD-98059, wortmannin, and LY-294002. A variety of specific antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate either the ethanol-induced contractions or the elevations of [Ca(2+)](i). Results of the present study support the suggestion that activation of tyrosine kinases (including the src family of cytoplasmic tyrosine kinases), PI-3Ks, and MAPK seems to play an important role in ethanol-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from rat aorta. These signaling pathways thus may be important in hypertension in human beings associated with chronic alcohol

  17. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.

    PubMed

    Pene, Frédéric; Claessens, Yann-Erick; Muller, Odile; Viguié, Franck; Mayeux, Patrick; Dreyfus, François; Lacombe, Catherine; Bouscary, Didier

    2002-09-26

    Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70(S6K)) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70(S6K) and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70(S6K) but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70(S6K). Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70(S6K) was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27(Kip1) protein. In the

  18. PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

    PubMed

    Desai, S; Pillai, P; Win-Piazza, H; Acevedo-Duncan, M

    2011-06-01

    The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

  19. Enhancement of morphological plasticity in hippocampal neurons by a physically modified saline via phosphatidylinositol-3 kinase.

    PubMed

    Roy, Avik; Modi, Khushbu K; Khasnavis, Saurabh; Ghosh, Supurna; Watson, Richard; Pahan, Kalipada

    2014-01-01

    Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer's disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.

  20. Integrin αvβ3 mediates the synergetic regulation of core-binding factor α1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling.

    PubMed

    Dai, Zhongquan; Guo, Feima; Wu, Feng; Xu, Hongjie; Yang, Chao; Li, Jinqiao; Liang, Peilong; Zhang, Hongyu; Qu, Lina; Tan, Yingjun; Wan, Yumin; Li, Yinghui

    2014-12-01

    Mechanical stimulation and biological factors coordinately regulate bone development and regeneration; however, the underlying mechanisms are poorly understood. Microgravity induces bone loss, which may be partly related to the development of resistance to local cytokines, including insulin-like growth factor 1 (IGF-1). Here, we report the involvement of integrin αvβ3 in microgravity-associated bone loss. An established OSE-3T3 cell model was stably transfected with a 6OSE2 (Osteoblast-Specific Element 2)-luciferase reporter and cultured under simulated microgravity (SMG) and hypergravity (HG) conditions in the presence or absence of IGF-1, the disintegrin echistatin, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or combinations of these agents. Activity of core-binding factor α1 (Cbfa1), an essential transcription factor for osteoblastic differentiation and osteogenesis, was reflected by luciferase activity. Different gravity conditions affected the induction of IGF-1 and subsequent effects on Cbfa1 transcription activity. SMG and HG influenced the expression and activity of integrin αvβ3 and phosphorylation level of p85. LY294002 inhibited the effects of HG or IGF-1 on Cbfa1 activity, indicating that HG and IGF-1 could increase Cbfa1 activity via PI3K signaling. Inhibition of integrin αvβ3 by echistatin attenuated the induction of IGF-1 and thus its effect on Cbfa1 activity under normal and HG conditions. Co-immunoprecipitation demonstrated that integrin β3 interacted with insulin receptor substrate 1, and that this interaction was decreased under SMG and increased under HG conditions. These results suggest that integrin αvβ3 mediates the synergetic regulation of Cbfa1 transcription activity by gravity and IGF-1 via PI3K signaling.

  1. Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH{sub 2}-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

    SciTech Connect

    Miyata, Yoshiki; Sato, Takashi . E-mail: satotak@ps.toyaku.ac.jp; Ito, Akira

    2005-11-04

    Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH{sub 2}-terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation.

  2. Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries

    SciTech Connect

    Keating, Aileen F.; Mark, Connie J.; Sen, Nivedita; Sipes, I. Glenn; Hoyer, Patricia B.

    2009-12-01

    4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 muM), or DMBA (1 muM), +- PI3 kinase inhibitor LY294002 (20 muM) or its inactive analog LY303511 (20 muM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P < 0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P < 0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P > 0.05) at any time, but did cause loss (P < 0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P < 0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P < 0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

  3. Differential sensitivities of trastuzumab (Herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors.

    PubMed

    Chan, Carmel T; Metz, Marianne Z; Kane, Susan E

    2005-05-01

    Her2 (erbB2/neu) is overexpressed in 25-30% of human breast cancers. Herceptin is a recombinant humanized Her2 antibody used to treat breast cancer patients with Her2 overexpression. Over a 5-month selection process, we isolated clones of BT474 (BT) human breast carcinoma cells (BT/Her(R)) that were resistant to Herceptin in vitro. In BT/Her(R) subclones, cell-surface, phosphorylated and total cellular Her2 protein remained high in the continuous presence of Herceptin. Likewise, the levels of cell-surface, phosphorylated, and total cellular Her3 and EGFR were either unchanged or only slightly elevated in BT/Her(R) subclones relative to BT cells. One BT/Her(R) subclone had substantially upregulated cell-surface EGFR, but this did not correlate with a higher relative resistance to Herceptin. In looking at the downstream PI-3K/Akt signaling pathway, phosphorylated and total Akt levels and Akt kinase activities were all sustained in BT/Her(R) subclones in the presence of Herceptin, but significantly downregulated in BT cells exposed to Herceptin. Whereas BT cells lost sensitivity to the PI-3K inhibitor LY294002 in the presence of Herceptin, BT/Her(R) subclones were equally sensitive to this agent in the presence and absence of Herceptin. This suggests that BT/Her(R) subclones acquired a Herceptin-resistant mechanism of PI-3K signaling. BT/Her(R) subclones were also sensitive to the EGFR kinase inhibitor AG1478 in the presence of Herceptin, to the same extent as BT cells. The BT/Her(R) subclones provide new insights into mechanisms of Herceptin resistance and suggest new treatment strategies in combination with other inhibitors targeted to signal transduction pathways.

  4. Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors.

    PubMed

    Singh, Abhay Kumar; Pantouris, Georgios; Borosch, Sebastian; Rojanasthien, Siripong; Cho, Thomas Yoonsang

    2017-01-01

    Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

  5. Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways

    PubMed Central

    Zwang, N. A.; Zhang, R.; Germana, S.; Fan, M. Y.; Hastings, W. D.; Cao, A.; Turka, L. A.

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4+ and CD8+ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4+ and CD8+ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4+ and CD8+ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity. PMID:27017850

  6. Class III phosphoinositide 3-kinase/VPS34 and dynamin are critical for apical endocytic recycling.

    PubMed

    Carpentier, Sarah; N'Kuli, Francisca; Grieco, Giuseppina; Van Der Smissen, Patrick; Janssens, Virginie; Emonard, Hervé; Bilanges, Benoît; Vanhaesebroeck, Bart; Gaide Chevronnay, Héloïse P; Pierreux, Christophe E; Tyteca, Donatienne; Courtoy, Pierre J

    2013-08-01

    Recycling is a limiting step for receptor-mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan-class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP-2, causing surface down-regulation. GFP-(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002-treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin-GFP and dependence of dynamin-GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K-III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.

  7. Inhibition of gap junctional Intercellular communication in WB-F344 rat liver epithelial cells by triphenyltin chloride through MAPK and PI3-kinase pathways

    PubMed Central

    2010-01-01

    Background Organotin compounds (OTCs) have been widely used as stabilizers in the production of plastic, agricultural pesticides, antifoulant plaints and wood preservation. The toxicity of triphenyltin (TPT) compounds was known for their embryotoxic, neurotoxic, genotoxic and immunotoxic effects in mammals. The carcinogenicity of TPT was not well understood and few studies had discussed the effects of OTCs on gap junctional intercellular communication (GJIC) of cells. Method In the present study, the effects of triphenyltin chloride (TPTC) on GJIC in WB-F344 rat liver epithelial cells were evaluated, using the scrape-loading dye transfer technique. Results TPTC inhibited GJIC after a 30-min exposure in a concentration- and time-dependent manner. Pre-incubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 and PI3K inhibitor LY294002 decreased substantially the inhibition of GJIC by TPTC. After WB-F344 cells were exposed to TPTC, phosphorylation of Cx43 increased as seen in Western blot analysis. Conclusions These results show that TPTC inhibits GJIC in WB-F344 rat liver epithelial cells by altering the Cx43 protein expression through both MAPK and PI3-kinase pathways. PMID:20591183

  8. A new selective AKT pharmacological inhibitor reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia cells.

    PubMed

    Martelli, A M; Tazzari, P L; Tabellini, G; Bortul, R; Billi, A M; Manzoli, L; Ruggeri, A; Conte, R; Cocco, L

    2003-09-01

    It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIP(L), and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-zeta and it was less

  9. Intracellular signal transduction pathways in the regulation of fowl sperm motility: evidence for the involvement of phosphatidylinositol 3-kinase (PI3-K) cascade.

    PubMed

    Ashizawa, Koji; Omura, Yusuke; Katayama, Seiichi; Tatemoto, Hideki; Narumi, Kazunori; Tsuzuki, Yasuhiro

    2009-07-01

    The possible role of PI3-K in the reversible temperature-dependent immobilization of fowl sperm motility was investigated by using PI3-K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3-K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40 degrees C but was maintained vigorously when the temperature was decreased to 30 degrees C. At 30 degrees C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl2 and 10 microM LY294002 or LY303511: around 70-80% of spermatozoa remained motile. In contrast, at 40 degrees C, the motility of spermatozoa was activated immediately after the addition of Ca(2+), but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca(2+)-supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca(2+) or LY303511 + Ca(2+) were almost the same values compared with those of Ca(2+) alone at 40 degrees C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility-regulating cascade. Immunoblotting of sperm extract using an antibody to PI3-K revealed a major cross-reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3-K. These results suggest that PI3-K may be positively involved in the calcium-regulated maintenance of flagellar movement of fowl spermatozoa at 40 degrees C.

  10. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

    PubMed Central

    Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation. PMID:28072855

  11. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

    PubMed

    Ohtsuka, Hiroko; Iguchi, Tomohiro; Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

  12. Carbamazepine enhances the activity of glutamate transporter type 3 via phosphatidylinositol 3-kinase.

    PubMed

    Lee, Gwanwoo; Huang, Yueming; Washington, Jacqueline M; Briggs, Nicole W; Zuo, Zhiyi

    2005-01-01

    Glutamate transporters (also called excitatory amino acid transporters, EAAT) participate in maintaining extracellular homeostasis of glutamate, a major excitatory neurotransmitter, and regulating glutamate neurotransmission. EAAT3, the major neuronal EAAT, may also regulate gamma-aminobutyric acid-mediated inhibitory neurotransmission. Dysfunction of EAAT3 has been shown to induce seizure in rats. We hypothesize that carbamazepine, a commonly used antiepileptic agent, enhances EAAT3 activity. We tested this hypothesis using oocytes artificially expressing EAAT3 and C6 rat glioma cells expressing endogenous EAAT3. In oocytes, carbamazepine dose-dependently enhanced EAAT3 activity. The EC50 of this carbamazepine effect was 12.2muM. The concentrations of carbamazepine to significantly enhance EAAT3 activity were within the therapeutic serum levels (17-51muM) of carbamazepine for the antiepileptic effect. Carbamazepine decreased the Km but did not change the maximal response of EAAT3 to glutamate. Carbamazepine-increased EAAT3 activity was inhibited by wortmannin or LY-294002, phosphatidylinositol 3-kinase (PI3K) inhibitors, but was not affected by staurosporine, chelerythrine or calphostin C, protein kinase C inhibitors. In C6 cells, carbamazepine also enhanced the endogenous EAAT3 activity. However, carbamazepine did not affect the activity of EAAT4 expressed in Cos7 cells. These results suggest that carbamazepine at clinically relevant concentrations specifically enhances the affinity of EAAT3 for glutamate to increase EAAT3 activity via a PI3K-dependent pathway. EAAT3 may be a therapeutic target for carbamazepine in the central nervous system.

  13. Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization.

    PubMed

    Zhuang, Zhi-Ye; Xu, Haoxing; Clapham, David E; Ji, Ru-Rong

    2004-09-22

    Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

  14. HspB8 mediates neuroprotection against OGD/R in N2A cells through the phosphoinositide 3-kinase/Akt pathway.

    PubMed

    Hu, Zhiping; Yang, Binbin; Mo, Xiaoye; Zhou, Fangfang

    2016-08-01

    In a previous study, we found that Heat shock protein B8 (HspB8) overexpression could prevent the apoptosis and reduced cell viability induced by OGD/R and showed that the neuroprotective effect of HspB8 was mediated by inhibition of the mitochondrial apoptotic pathway. In recent study, HspB8 has been shown to protect the heart against ischemia/reperfusion (I/R) injury via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, whether this protective effect applied to brain I/R injury remained unexplored. To further test the mechanism of HspB8's effects in brain, we used oxygen-glucose deprivation followed by reperfusion (OGD/R), an in vitro model of ischemia to examine the involvement of PI3K/Akt signaling by treating mouse neuroblastoma cells (N2A cells) (untransfected or transfected with an HspB8 expression vector) with the PI3K inhibitor LY294002 before OGD/R. Our results revealed that the apoptosis-suppressing effect of HspB8 was mediated by the PI3K/Akt pathway. Therefore, HspB8 protected the N2A cells against OGD/R insult, possibly by activating the PI3K/Akt signaling pathway.

  15. Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation

    PubMed Central

    Liu, Shan-Lu; Lerman, Michael I.; Miller, A. Dusty

    2003-01-01

    Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway. PMID:12829832

  16. Phosphatidylinositol 3-kinase/Akt signaling pathway mediates acupuncture-induced dopaminergic neuron protection and motor function improvement in a mouse model of Parkinson's disease.

    PubMed

    Kim, Seung-Nam; Kim, Seung-Tae; Doo, Ah-Reum; Park, Ji-Yeun; Moon, Woongjoon; Chae, Younbyoung; Yin, Chang Shik; Lee, Hyejung; Park, Hi-Joon

    2011-10-01

    It has been reported that acupuncture treatment reduced dopaminergic neuron degeneration in Parkinson's disease (PD) models. However, the mechanistic pathways underlying, such neuroprotection, are poorly understood. Here, we investigated the effects and the underlying mechanism of acupuncture in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). First, we observed that MPTP-induced impairment of Akt activation, but not MPTP-induced c-Jun activation, was effectively restored by acupuncture treatment in the substantia nigra. Furthermore, we demonstrated for the first time that the brain-specific blockade of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, by intranasal administration of LY294002, a specific inhibitor of PI3K/Akt signaling pathway, significantly blocked acupuncture-induced dopaminergic neuron protection and motor function improvement. Our results provide evidence that PI3K/Akt signaling pathway may play a central role in the mechanism underlying acupuncture-induced benefits in Parkinsonian mice.

  17. Mitogen-activated protein kinases and phosphatidylinositol 3-kinase are involved in Prevotella intermedia-induced proinflammatory cytokines expression in human periodontal ligament cells.

    PubMed

    Guan, Su-Min; Zhang, Ming; He, Jian-Jun; Wu, Jun-Zheng

    2009-08-28

    Chronic periodontitis is an inflammatory disease affecting periodontal connective tissues and alveolar bone. Proinflammatory mediators induced by periodontal pathogens play vital roles in the initiation and progression of the disease. In this study, we examined whether Prevotella intermedia induces proinflammatory cytokines expression in human periodontal ligament cells (hPDLs). The mRNA expression and protein production were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) respectively. P. intermedia treatment dose- and time-dependently increased IL-6, IL-8 and M-CSF, but not IL-1beta and TNF-alpha mRNA expression and protein secretion. Preincubation of hPDLs with extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 kinase and phosphatidylinositol 3-kinase (PI3K) inhibitors PD98059, SP600125, SB203580 and LY294002 resulted in significant reduction in P. intermedia-induced IL-6, IL-8 and M-CSF expression. Blocking the synthesis of prostaglandin E(2) (PGE(2)) by indomethacin also abolished the stimulatory effects of P. intermedia on cytokines expression. Our results indicate that P. intermedia induces proinflammatory cytokines through MAPKs and PI3K signaling pathways, and PGE(2) is involved in the P. intermedia-induced proinflammatory cytokines upregulation.

  18. Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo.

    PubMed Central

    Vanhaesebroeck, B; Higashi, K; Raven, C; Welham, M; Anderson, S; Brennan, P; Ward, S G; Waterfield, M D

    1999-01-01

    Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules. PMID:10064595

  19. 17β-estradiol impedes Bax-involved mitochondrial apoptosis of retinal nerve cells induced by oxidative damage via the phosphatidylinositol 3-kinase/Akt signal pathway.

    PubMed

    Li, Hongbo; Wang, Baoying; Zhu, Chunhui; Feng, Yan; Wang, Shaolan; Shahzad, Muhammad; Hu, Chenghu; Mo, Mingshu; Du, Fangying; Yu, Xiaorui

    2013-07-01

    Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17β-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H(2)O(2) could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H(2)O(2)-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H(2)O(2)-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H(2)O(2). In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H(2)O(2). And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H(2)O(2) damage were significantly retrieved by LY294002 administration. Taken

  20. Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    PubMed Central

    Hussain, Azhar R.; Ahmed, Saeeda O.; Ahmed, Maqbool; Khan, Omar S.; Al AbdulMohsen, Sally; Platanias, Leonidas C.; Al-Kuraya, Khawla S.; Uddin, Shahab

    2012-01-01

    Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways. PMID:22768179

  1. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    PubMed Central

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

  2. Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity.

    PubMed

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.

  3. Involvement of PI 3 kinase/Akt-dependent Bad phosphorylation in Toxoplasma gondii-mediated inhibition of host cell apoptosis.

    PubMed

    Quan, Juan-Hua; Cha, Guang-Ho; Zhou, Wei; Chu, Jia-Qi; Nishikawa, Yoshifumi; Lee, Young-Ha

    2013-04-01

    Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.

  4. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

    PubMed

    Koushik, Amrita B; Welter, Brenda H; Rock, Michelle L; Temesvari, Lesly A

    2014-03-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

  5. Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

    SciTech Connect

    Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H.

    2012-09-17

    A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

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

    PubMed

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

    2015-11-01

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

  7. Static magnetic field enhances the viability and proliferation rate of adipose tissue-derived mesenchymal stem cells potentially through activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway.

    PubMed

    Marędziak, Monika; Tomaszewski, Krzysztof; Polinceusz, Paulina; Lewandowski, Daniel; Marycz, Krzysztof

    2017-01-01

    The aim of this work was to investigate the effects of 0.5T static magnetic field (sMF) on the viability and proliferation rate of human adipose-derived mesenchymal stromal stem cells (hASCs) via activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathway. In a 7-d culture we examined cell growth kinetic and population doubling time (PDT). We also examined cell morphology and the cellular senescence markers level. Exposure to sMF enhanced the viability of these cells. However, the effect was blocked by treating the cells with LY294002, a P13K inhibitor. We compared this effect by Western Blot analysis of Akt protein expression. We also examined whether the cell response on sMF stimulation is dependent on integrin engagement and we measured integrin gene expression. Our results suggest that stimulation using sMF is a viable method to improve hASC viability. sMF is involved in mechanisms associated with controlling cell proliferative potential signaling events.

  8. PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease.

    PubMed

    Quesada, Arnulfo; Lee, Becky Y; Micevych, Paul E

    2008-04-01

    Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.

  9. Dexmedetomidine increases the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes: the involvement of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Do, Sang-Hwan; Park, Seong-Joo; Shin, Hyun-Jung; Paik, Hye-Sun; Zuo, Zhiyi; Yoon, Hea-Jo; Ryu, Jung-Hee

    2014-09-05

    Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30 μM l-glutamate in the presence of 0.1-30 nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.

  10. Promotion of melanoma cell invasion and tumor metastasis by microcystin-LR via phosphatidylinositol 3-kinase/AKT pathway.

    PubMed

    Xu, Pengfei; Zhang, Xu-Xiang; Miao, Chen; Fu, Ziyi; Li, Zhengrong; Zhang, Gen; Zheng, Maqing; Liu, Yuefang; Yang, Liuyan; Wang, Ting

    2013-08-06

    Recently, we have indicated that microcystin-LR, a cyanobacterial toxin produced in eutrophic lakes or reservoirs, can increase invasive ability of melanoma MDA-MB-435 cells; however, the stimulatory effect needs identification by in vivo experiment and the related molecular mechanism is poorly understood. In this study, in vitro and in vivo experiments were conducted to investigate the effect of microcystin-LR on invasion and metastasis of human melanoma cells, and the underlying molecular mechanism was also explored. MDA-MB-435 xenograft model assay showed that oral administration of nude mice with microcystin-LR at 0.001-0.1 mg/kg/d posed no significant effect on tumor weight. Histological examination demonstrated that microcystin-LR could promote lung metastasis, which is confirmed by Matrigel chamber assay suggesting that microcystin-LR treatment at 25 nM can increase the invasiveness of MDA-MB-435 cells. In vitro and in vivo experiments consistently showed that microcystin-LR exposure increased mRNA and protein levels of matrix metalloproteinases (MMP-2/-9) by activating phosphatidylinositol 3-kinase (PI3-K)/AKT. Additionally, microcystin-LR treatment at low doses (≤25 nM) decreased lipid phosphatase PTEN expression, and the microcystin-induced invasiveness enhancement and MMP-2/-9 overexpression were reversed by the PI3-K/AKT chemical inhibitor LY294002 and AKT siRNA, indicating that microcystin-LR promotes invasion and metastasis of MDA-MB-435 cells via the PI3-K/AKT pathway.

  11. Induction of granzyme B expression in T-cell receptor/CD28-stimulated human regulatory T cells is suppressed by inhibitors of the PI3K-mTOR pathway

    PubMed Central

    2009-01-01

    Background Regulatory T cells (Tregs) can employ a cell contact- and granzyme B-dependent mechanism to mediate suppression of bystander T and B cells. Murine studies indicate that granzyme B is involved in the Treg-mediated suppression of anti-tumor immunity in the tumor microenvironment and in the Treg-mediated maintenance of allograft survival. In spite of its central importance, a detailed study of granzyme B expression patterns in human Tregs has not been performed. Results Our data demonstrated that natural Tregs freshly isolated from the peripheral blood of normal adults lacked granzyme B expression. Tregs subjected to prolonged TCR and CD28 triggering, in the presence of IL-2, expressed high levels of granzyme B but CD3 stimulation alone or IL-2 treatment alone failed to induce granzyme B. Treatment of Tregs with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin or the PI3 kinase (PI3K) inhibitor LY294002 markedly suppressed granzyme B expression. However, neither rapamycin, as previously reported by others, nor LY294002 inhibited Treg proliferation or induced significant cell death in TCR/CD28/IL-2 stimulated cells. The proliferation rate of Tregs was markedly higher than that of CD4+ conventional T cells in the setting of rapamycin treatment. Tregs expanded by CD3/CD28/IL-2 stimulation without rapamycin demonstrated increased in vitro cytotoxic activity compared to Tregs expanded in the presence of rapamycin in both short term (6 hours) and long term (48 hours) cytotoxicity assays. Conclusion TCR/CD28 mediated activation of the PI3K-mTOR pathway is important for granyzme B expression but not proliferation in regulatory T cells. These findings may indicate that suppressive mechanisms other than granzyme B are utilized by rapamycin-expanded Tregs. PMID:19930596

  12. The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High-Throughput Cell-Based Screens

    DTIC Science & Technology

    2005-02-01

    cells. Psycho- Calmodulin antagonists inhibit insulin -stimulated GLUT4 ( glucose trans- pharmacology (Berl.) 150, 383-390. porter 4) translocation by...AD Award Number: W81XWH-04-1-0169 TITLE: The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High ...Phosphoinositide-3-Kinase Pathway Through High -Throughput Cell-Based Screens 6. AUTHOR(S) William R. Sellers, M.D. 7. PERFORMING ORGANIZA TION NAME(S) AND

  13. Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.

    PubMed

    Miller, Michelle S; Pinson, Jo-Anne; Zheng, Zhaohua; Jennings, Ian G; Thompson, Philip E

    2013-02-01

    Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.

  14. Investigation into the Role of PI3K and JAK3 Kinase Inhibitors in Murine Models of Asthma.

    PubMed

    Wagh, Akshaya D; Sharma, Manoranjan; Mahapatra, Jogeshwar; Chatterjee, Abhijeet; Jain, Mukul; Addepalli, Veeranjaneyulu

    2017-01-01

    Asthma is a clinical disorder commonly characterized by chronic eosinophilic inflammation, remodeling and hyper responsiveness of the airways. However, the kinases like Phosphoinositide 3 kinase (PI3K) and Janus kinase 3 (JAK3) are involved in mast cell proliferation, activation, recruitment, migration, and prolonged survival of inflammatory cells. The present study was designed to evaluate the in-vivo comparative effects of two kinase inhibitors on airway inflammation and airway remodeling in acute and chronic models of asthma. Mice were sensitized twice intra-peritoneally and then challenged by inhalation with ovalbumin (OVA). They developed an extensive inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening similar to pathologies observed in human asthma. The effects of PI3K inhibitor (30 mg/kg, p.o), JAK3 inhibitor (30 mg/kg, p.o) and Dexamethasone (0.3 mg/kg) on airway inflammation and remodeling in OVA sensitized/challenged BALB/c mice were evaluated. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage (BAL) and histological examinations were carried out. It was observed that kinase inhibitors significantly reduced airway inflammation as evidenced by the decrease in pro inflammatory cytokines in BALF and lung homogenate and inflammatory cell count in sensitized mice after allergen challenge. Lung histological analysis showed increased infiltration of inflammatory cells, hyperplasia of goblet cells and the collagen deposition, which were significantly reduced with kinase inhibitor. In conclusion, our data suggest that PI3K and JAK3 inhibitors showed promising alternative therapeutic activity in asthma, which might significantly counteract the airway inflammation in patients with allergic asthma.

  15. Investigation into the Role of PI3K and JAK3 Kinase Inhibitors in Murine Models of Asthma

    PubMed Central

    Wagh, Akshaya D.; Sharma, Manoranjan; Mahapatra, Jogeshwar; Chatterjee, Abhijeet; Jain, Mukul; Addepalli, Veeranjaneyulu

    2017-01-01

    Asthma is a clinical disorder commonly characterized by chronic eosinophilic inflammation, remodeling and hyper responsiveness of the airways. However, the kinases like Phosphoinositide 3 kinase (PI3K) and Janus kinase 3 (JAK3) are involved in mast cell proliferation, activation, recruitment, migration, and prolonged survival of inflammatory cells. The present study was designed to evaluate the in-vivo comparative effects of two kinase inhibitors on airway inflammation and airway remodeling in acute and chronic models of asthma. Mice were sensitized twice intra-peritoneally and then challenged by inhalation with ovalbumin (OVA). They developed an extensive inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening similar to pathologies observed in human asthma. The effects of PI3K inhibitor (30 mg/kg, p.o), JAK3 inhibitor (30 mg/kg, p.o) and Dexamethasone (0.3 mg/kg) on airway inflammation and remodeling in OVA sensitized/challenged BALB/c mice were evaluated. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage (BAL) and histological examinations were carried out. It was observed that kinase inhibitors significantly reduced airway inflammation as evidenced by the decrease in pro inflammatory cytokines in BALF and lung homogenate and inflammatory cell count in sensitized mice after allergen challenge. Lung histological analysis showed increased infiltration of inflammatory cells, hyperplasia of goblet cells and the collagen deposition, which were significantly reduced with kinase inhibitor. In conclusion, our data suggest that PI3K and JAK3 inhibitors showed promising alternative therapeutic activity in asthma, which might significantly counteract the airway inflammation in patients with allergic asthma. PMID:28293189

  16. Efficacy of Phosphatidylinositol-3 Kinase Inhibitors in a Primary Mouse Model of Undifferentiated Pleomorphic Sarcoma

    PubMed Central

    Kim, Suzy; Dodd, Rebecca D.; Mito, Jeffrey K.; Ma, Yan; Kim, Yongbaek; Riedel, Richard F.; Kirsch, David G.

    2012-01-01

    Recent advances in sarcoma genomics have identified novel mutations in the PI3K pathway in human sarcomas. Here, we use a mouse model of primary soft-tissue sarcoma for preclinical testing of doxorubicin and inhibitors of the PI3K pathway: BKM120 (PI3K inhibitor) and BEZ235 (a dual PI3K/mTOR inhibitor). Doxorubicin-treated tumors (n = 15) showed a partial response rate of 6.6%, just as the majority of human sarcomas do not respond to doxorubicin. Treatment with BKM120 elicited a partial response in 50% of tumors (n = 10), which was also seen in combination with doxorubicin (n = 10). Additionally, BKM120 treatment produced a robust delay in tumor growth kinetics. BEZ235-treated tumors (n = 9) showed a complete response rate of 11.1%. Combining BEZ235 with doxorubicin (n = 10) increased the complete response rate to 50% (P = 0.035). These studies demonstrate that PI3K pathway inhibition is a viable and attractive target for soft-tissue sarcomas. PMID:22619567

  17. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

    SciTech Connect

    Singh, Alok R.; Peirce, Susan K.; Joshi, Shweta; Durden, Donald L.

    2014-09-10

    -3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo. - Highlights: • First genetic evidence that PTEN controls LPS/TLR4 signaling in B lymphocytes. • Evidence that PTEN regulates LPS induced lymphoproliferation in vivo. • PI-3 kinase inhibitors block LPS induced lymphoproliferation in vivo.

  18. Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.

    PubMed Central

    Yamada, Momoko; Banno, Yoshiko; Takuwa, Yoh; Koda, Masahiro; Hara, Akira; Nozawa, Yoshinori

    2004-01-01

    To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cd. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cd cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways. PMID:14640974

  19. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

    PubMed

    Göckeritz, Elisa; Kerwien, Susan; Baumann, Michael; Wigger, Marion; Vondey, Verena; Neumann, Lars; Landwehr, Thomas; Wendtner, Clemens M; Klein, Christian; Liu, Ningshu; Hallek, Michael; Frenzel, Lukas P; Krause, Günter

    2015-11-01

    Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

  20. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    SciTech Connect

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; Barros, Tiago; Kuriyan, John; Boggon, Titus J.

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

  1. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    DOE PAGES

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; ...

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain ofmore » FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.« less

  2. Capitalizing on tumor genotyping: Towards the design of mutation specific inhibitors of phosphoinsitide-3-kinase

    SciTech Connect

    Gabelli, Sandra B.; Duong-Ly, Krisna C.; Brower, Evan T.; Amzel, L. Mario

    2011-09-06

    PI3Ks catalyze the phosphorylation of the inositol hydroxyls of phosphoinositide membrane components. The changes in phosphorylation of the inositides recruit proteins to the plasma membrane that initiate important signaling cascades. PI3K{alpha}, one of the class IA PI3Ks, is highly mutated in cancers. All mutations analyzed result in an increase in enzymatic activity. The structures of this enzyme determined by X-ray diffraction, provide a framework for analyzing the possible structural effect of these mutations and their effect on the enzymatic activity. Many of the mutations occur at domain interfaces where they can affect domain interactions and relieve the inhibition of the wild-type enzyme by the nSH2 domain of p85. This mechanism is analogous to the mechanism of physiological activation by activated tyrosine-kinase receptors in which the phosphorylated tyrosine of the receptor (or their substrates) dislodges the nSH2 from its inhibitory position in the complex by competing with its binding to a loop in the helical domain. Other mutations in the kinase domain can directly affect the conformation of the catalytic site. One mutation, His1047Arg, uses a completely different mechanism: it changes the conformation of the C-terminal loop in such a way that it increases the interaction of the enzyme with the membrane, granting increased access to the phosphoinositide substrates. Taking advantage of the reliance of some cancers on the increased activity of mutated PI3K{alpha}, will require the development of isoform-specific, mutant-specific inhibitors. The structural, biochemical and physiological data that are becoming available for PI3Ks are an important first step in this direction.

  3. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    PubMed Central

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes. PMID:8611143

  4. Transduction of Growth or Mitogenic Signals into Translational Activation of TOP mRNAs Is Fully Reliant on the Phosphatidylinositol 3-Kinase-Mediated Pathway but Requires neither S6K1 nor rpS6 Phosphorylation

    PubMed Central

    Stolovich, Miri; Tang, Hua; Hornstein, Eran; Levy, Galit; Cohen, Ruth; Bae, Sun Sik; Birnbaum, Morris J.; Meyuhas, Oded

    2002-01-01

    Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Bα (PKBα). Likewise, overexpression of constitutively active PI3-kinase or PKBα can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway. PMID:12417714

  5. Protective effects of statins on L-DOPA neurotoxicity due to the activation of phosphatidylinositol 3-kinase and free radical scavenging in PC12 cell culture.

    PubMed

    Koh, Seong-Ho; Park, Hyun-Hee; Choi, Na-Young; Lee, Kyu-Yong; Kim, Sangjae; Lee, Young Joo; Kim, Hee-Tae

    2011-01-25

    Neurotoxic effects have been suggested for l-3,4-dihydroxyphenylalanine (L-DOPA), while neuroprotective effects have been proposed for statins. We investigated whether certain statins (simvastatin or pitavastatin) could inhibit L-DOPA neurotoxicity. Neuronally-differentiated PC12 (nPC12) cells were treated with L-DOPA and/or statins for 24h, and their viabilities were measured using a cell counting kit, trypan blue staining, and 4',6-diamidino-2-phenylindole (DAPI) staining. Free radical and specific intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. High concentrations of l-DOPA reduced nPC12 cell viability, but combined treatment with statins restored viability. Treatment with 200 μM L-DOPA increased free radical and hydroxyl radical levels, but combined treatment with 5 μM statins decreased these levels. Survival-related signaling proteins were decreased in nPC12 cells treated with 200 μM L-DOPA, but combined treatment with 5μM statins significantly increased the levels of these proteins. Treatment with 200 μM L-DOPA significantly increased death-related signaling proteins, while combined treatment with 5 μM statins reduced the levels of these proteins. Pretreatment with LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, before combined treatment with statins and L-DOPA almost completely blocked the protective effects of statins. These results indicate that statins reduce L-DOPA neurotoxicity by lowering oxidative stress and by enhancing survival signals and inhibiting death signals via activation of the PI3K pathway.

  6. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML.

    PubMed

    Park, S; Chapuis, N; Bardet, V; Tamburini, J; Gallay, N; Willems, L; Knight, Z A; Shokat, K M; Azar, N; Viguié, F; Ifrah, N; Dreyfus, F; Mayeux, P; Lacombe, C; Bouscary, D

    2008-09-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways are frequently activated in acute myelogenous leukemia (AML). mTORC1 inhibition with RAD001 induces PI3K/Akt activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. PI-103 is a new potent PI3K/Akt and mTOR inhibitor. In human leukemic cell lines and in primary blast cells from AML patients, PI-103 inhibited constitutive and growth factor-induced PI3K/Akt and mTORC1 activation. PI-103 was essentially cytostatic for cell lines and induced cell cycle arrest in the G1 phase. In blast cells, PI-103 inhibited leukemic proliferation, the clonogenicity of leukemic progenitors and induced mitochondrial apoptosis, especially in the compartment containing leukemic stem cells. In contrast, apoptosis was not induced with RAD001 and IC87114 association, which specifically inhibits mTORC1 and p110delta activity, respectively. PI-103 had additive proapoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, PI-103 did not induce apoptosis in normal CD34(+) cells and had moderate effects on their clonogenic and proliferative properties. Here, we demonstrate that multitargeted therapy against PI3K/Akt and mTOR with PI-103 may be of therapeutic value in AML.

  7. Puquitinib mesylate, an inhibitor of phosphatidylinositol 3-kinase p110δ, for treating relapsed or refractory non-Hodgkin's lymphoma

    PubMed Central

    Zhan, Jing; Xia, Yi; Sun, Peng; Bi, Xi-Wen; Liu, Pan-Pan; Li, Zhi-Ming; Li, Su; Zou, Ben-Yan; Jiang, Wen-Qi

    2015-01-01

    Objectives To determine the safety of Puquitinib Mesylate (XC-302), an oral inhibitor of phosphatidylinositol 3-kinase, in treating relapsed or refractory non-Hodgkin's lymphoma (NHL). Methods Between October 2013 and July 2015, 21 patients from Sun Yat-sen University Cancer Center were treated twice daily on each day of a 28-day cycle (median number of cycles, 2; maximum, 20) with XC-302 at a post prandial dose of 25 mg, 37.5 mg, or 50 mg. Adverse events (AEs), AUClast and Cmax, response rates, and overall survival were assessed. Results Patients had received a median (range) of 1 (1 to 3) previous cancer treatments. At the latest follow-up, two patients were still benefitting from the study. The most common drug-related AEs were elevations in alanine transaminase (ALT, 14 of 21 patients) and aspartate transaminase (AST, 7 of 21 patients). Four patients, both in the-50-mg group, had dose-limiting toxicities, and therapy was discontinued in a fifth because of persistent abnormal liver function. The overall response rate was 2 of19. Serum concentrations of XC-302 increased in a dose-dependent pattern. Median progression-free survival in all patients was 1.9 (95% CI, 1.7 to 2.0) months. Conclusion XC-302 has an acceptable safety profile and offers potential therapeutic value to patients with relapsed or refractory non-Hodgkin lymphoma. PMID:26510909

  8. The novel PI3 kinase inhibitor, BAY 80-6946, impairs melanoma growth in vivo and in vitro.

    PubMed

    Schneider, Philine; Schön, Margarete; Pletz, Nadin; Seitz, Cornelia S; Liu, Ningshu; Ziegelbauer, Karl; Zachmann, Karolin; Emmert, Steffen; Schön, Michael P

    2014-08-01

    Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.

  9. Pulmonary administration of phosphoinositide 3-kinase inhibitor is a curative treatment for chronic obstructive pulmonary disease by alveolar regeneration.

    PubMed

    Horiguchi, Michiko; Oiso, Yuki; Sakai, Hitomi; Motomura, Tomoki; Yamashita, Chikamasa

    2015-09-10

    Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causing widespread and irreversible alveoli collapse. The discovery of a low-molecular-weight compound that induces regeneration of pulmonary alveoli is of utmost urgency to cure intractable pulmonary diseases such as COPD. However, a practically useful compound for regenerating pulmonary alveoli is yet to be reported. Previously, we have elucidated that Akt phosphorylation is involved in a differentiation-inducing molecular mechanism of human alveolar epithelial stem cells, which play a role in regenerating pulmonary alveoli. In the present study, we directed our attention to phosphoinositide 3-kinase (PI3K)-Akt signaling and examined whether PI3K inhibitors display the pulmonary alveolus regeneration. Three PI3K inhibitors with different PI3K subtype specificities (Wortmannin, AS605240, PIK-75 hydrochloride) were tested for the differentiation-inducing effect on human alveolar epithelial stem cells, and Wortmannin demonstrated the most potent differentiation-inducing activity. We evaluated Akt phosphorylation in pulmonary tissues of an elastase-induced murine COPD model and found that Akt phosphorylation in the pulmonary tissue was enhanced in the murine COPD model compared with normal mice. Then, the alveolus-repairing effect of pulmonary administration of Wortmannin to murine COPD model was evaluated using X-ray CT analysis and hematoxylin-eosin staining. As a result, alveolar damages were repaired in the Wortmannin-administered group to a similar level of normal mice. Furthermore, pulmonary administration of Wortmannin induced a significant recovery of the respiratory function, compared to the control group. These results indicate that Wortmannin is capable of inducing differentiation of human alveolar epithelial stem cells and represents a promising drug candidate for curative treatment of pulmonary alveolar destruction in COPD.

  10. Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

    SciTech Connect

    Van Aller, Glenn S.; Carson, Jeff D.; Tang, Wei; Peng, Hao; Zhao, Lin; Copeland, Robert A.; Tummino, Peter J.; Luo, Lusong

    2011-03-11

    Research highlights: {yields} Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. {yields} EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. {yields} Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. {yields} These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K{sub i} values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

  11. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

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

    PubMed

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

    2015-12-01

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

  13. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment.

    PubMed

    Singh, Alok R; Peirce, Susan K; Joshi, Shweta; Durden, Donald L

    2014-09-10

    Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN(fl/fl) mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3

  14. Invention of a novel photodynamic therapy for tumors using a photosensitizing PI3K inhibitor.

    PubMed

    Hayashida, Yushi; Ikeda, Yuka; Sawada, Koichi; Kawai, Katsuhisa; Kato, Takuma; Kakehi, Yoshiyuki; Araki, Nobukazu

    2016-08-01

    XL147 (SAR245408, pilaralisib), an ATP-competitive pan-class I phosphoinositide 3-kinase (PI3K) inhibitor, is a promising new anticancer drug. We examined the effect of the PI3K inhibitor on PC3 prostate cancer cells under a fluorescence microscope and found that XL147-treated cancer cells are rapidly injured by blue wavelength (430 nm) light irradiation. During the irradiation, the cancer cells treated with 0.2-2 μM XL147 showed cell surface blebbing and cytoplasmic vacuolation and died within 15 min. The extent of cell injury/death was dependent on the dose of XL147 and the light power of the irradiation. These findings suggest that XL147 might act as a photosensitizing reagent in photodynamic therapy (PDT) for cancer. Moreover, the cytotoxic effect of photosensitized XL147 was reduced by pretreatment with other ATP-competitive PI3K inhibitors such as LY294002, suggesting that the cytotoxic effect of photosensitized XL147 is facilitated by binding to PI3K in cells. In a single-cell illumination analysis using a fluorescent probe to identify reactive oxygen species (ROS), significantly increased ROS production was observed in the XL147-treated cells when the cell was illuminated with blue light. Taken together, it is conceivable that XL147, which is preferentially accumulated in cancer cells, could be photosensitized by blue light to produce ROS to kill cancer cells. This study will open up new possibilities for PDT using anticancer drugs.

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

    PubMed

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

    2007-03-01

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

  16. Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction.

    PubMed

    Sánchez, María G; Ruiz-Llorente, Lidia; Sánchez, Ana M; Díaz-Laviada, Inés

    2003-09-01

    Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.

  17. Progesterone increases brain-derived neuroptrophic factor expression and protects against glutamate toxicity in a mitogen-activated protein kinase- and phosphoinositide-3 kinase-dependent manner in cerebral cortical explants.

    PubMed

    Kaur, Paramjit; Jodhka, Parmeet K; Underwood, Wendy A; Bowles, Courtney A; de Fiebre, Nancyellen C; de Fiebre, Christopher M; Singh, Meharvan

    2007-08-15

    The higher prevalence and risk for Alzheimer's disease in women relative to men has been partially attributed to the precipitous decline in gonadal hormone levels that occurs in women following the menopause. Although considerable attention has been focused on the consequence of estrogen loss, and thus estrogen's neuroprotective potential, it is important to recognize that the menopause results in a precipitous decline in progesterone levels as well. In fact, progesterone is neuroprotective, although the precise mechanisms involved remain unclear. Based on our previous observation that progesterone elicits the phosphorylation of ERK and Akt, key effectors of the neuroprotective mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3-K) pathways, respectively, we determined whether activation of either of these pathways was necessary for progesterone-induced protection. With organotypic explants (slice culture) of the cerebral cortex, we found that progesterone protected against glutamate-induced toxicity. Furthermore, these protective effects were inhibited by either the MEK1/2 inhibitor UO126 or the PI3-K inhibitor LY294002, supporting the requirement for both the MAPK and PI3-K pathways in progesterone-induced protection. In addition, at a concentration and duration of treatment consistent with our neuroprotection data, progesterone also increased the expression of brain-derived neurotrophic factor (BDNF), at the level of both protein and mRNA. This induction of BDNF may be relevant to the protective effects of progesterone, in that inhibition of Trk signaling, with K252a, inhibited the protective effects of progesterone. Collectively, these data suggest that progesterone is protective via multiple and potentially related mechanisms. (c) 2007 Wiley-Liss, Inc.

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

    PubMed

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

    2010-11-24

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

  19. Combining TRAIL with PI3 Kinase or HSP90 inhibitors enhances apoptosis in colorectal cancer cells via suppression of survival signaling

    PubMed Central

    Saturno, Grazia; Valenti, Melanie; De Haven Brandon, Alexis; Thomas, George V.; Eccles, Suzanne; Clarke, Paul A.; Workman, Paul

    2013-01-01

    TRAIL has been shown to induce apoptosis in cancer cells, but in some cases they fail to respond to this ligand. We explored the ability of representative phosphatidylinositol-3-kinase (PI3 Kinase)/mTOR and HSP90 inhibitors to overcome TRAIL resistance by increasing apoptosis in colorectal cancer models. We determined the sensitivity of 27 human colorectal cancer and 2 non-transformed colon epithelial cell lines to TRAIL treatment. A subset of the cancer cell lines with a range of responses to TRAIL was selected from the panel for treatment with TRAIL combined with the PI3 Kinase/mTOR inhibitor PI-103 or the HSP90 inhibitor 17-AAG (tanespimycin). Two TRAIL-resistant cell lines were selected for in vivo combination studies with TRAIL and 17-AAG. We found that 13 colorectal cancer cell lines and the 2 non-transformed colon epithelial cell lines were resistant to TRAIL. We demonstrated that co-treatment of TRAIL and PI-103 or 17-AAG was synergistic or additive and significantly enhanced apoptosis in colorectal cancer cells. This was associated with decreased expression or activity of survival protein biomarkers such as ERBB2, AKT, IKKα and XIAP. In contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We show here for the first time that co-treatment in vivo with TRAIL and 17-AAG in two TRAIL-resistant human colorectal cancer xenograft models resulted in significantly greater tumor growth inhibition compared to single treatments. We propose that combining TRAIL with PI3 Kinase/mTOR or HSP90 inhibitors has therapeutic potential in the treatment of TRAIL-resistant colorectal cancers. PMID:23852390

  20. Molecular modeling study of CP-690550 derivatives as JAK3 kinase inhibitors through combined 3D-QSAR, molecular docking, and dynamics simulation techniques.

    PubMed

    Wang, Jing Li; Cheng, Li Ping; Wang, Tian Chi; Deng, Wei; Wu, Fan Hong

    2017-03-01

    To develop more potent JAK3 kinase inhibitors, a series of CP-690550 derivatives were investigated using combined molecular modeling techniques, such as 3D-QSAR, molecular docking and molecular dynamics (MD). The leave-one-out correlation (q(2)) and non-cross-validated correlation coefficient (r(2)) of the best CoMFA model are 0.715 and 0.992, respectively. The q(2) and r(2) values of the best CoMSIA model are 0.739 and 0.995, respectively. The steric, electrostatic, and hydrophobic fields played important roles in determining the inhibitory activity of CP-690550 derivatives. Some new JAK3 kinase inhibitors were designed. Some of them have better inhibitory activity than the most potent Tofacitinib (CP-690550). Molecular docking was used to identify some key amino acid residues at the active site of JAK3 protein. 10ns MD simulations were successfully performed to confirm the detailed binding mode and validate the rationality of docking results. The calculation of the binding free energies by MMPBSA method gives a good correlation with the predicted biological activity. To our knowledge, this is the first report on MD simulations and free energy calculations for this series of compounds. The combination results of this study will be valuable for the development of potent and novel JAK3 kinase inhibitors.

  1. Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells.

    PubMed

    Ciarcia, Roberto; Damiano, Sara; Montagnaro, Serena; Pagnini, Ugo; Ruocco, Antonio; Caparrotti, Giuseppe; d'Angelo, Danila; Boffo, Silvia; Morales, Fátima; Rizzolio, Flavio; Florio, Salvatore; Giordano, Antonio

    2013-09-01

    Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca (2+)-ATPase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant disease.

  2. Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

    PubMed

    Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

    2012-01-01

    Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients.

  3. Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

    NASA Technical Reports Server (NTRS)

    Pavalko, Fredrick M.; Gerard, Rita L.; Ponik, Suzanne M.; Gallagher, Patricia J.; Jin, Yijun; Norvell, Suzanne M.

    2003-01-01

    In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. Copyright 2002 Wiley-Liss, Inc.

  4. A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

    PubMed

    Wang, Liqin; Šuštić, Tonći; Leite de Oliveira, Rodrigo; Lieftink, Cor; Halonen, Pasi; van de Ven, Marieke; Beijersbergen, Roderick L; van den Heuvel, Michel M; Bernards, René; van der Heijden, Michiel S

    2017-01-17

    Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

  5. Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway

    PubMed Central

    Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D.; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn

    2017-01-01

    Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted. PMID:28107519

  6. Progress in the Preclinical Discovery and Clinical Development of Class I and Dual Class I/IV Phosphoinositide 3-Kinase (PI3K) Inhibitors

    PubMed Central

    Shuttleworth, S.J; Silva, F.A; Cecil, A.R.L; Tomassi, C.D; Hill, T.J; Raynaud, F.I; Clarke, P.A; Workman, P

    2011-01-01

    The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer. PMID:21649578

  7. Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach.

    PubMed

    Niedermeier, Matthias; Hennessy, Bryan T; Knight, Zachary A; Henneberg, Marina; Hu, Jianhua; Kurtova, Antonina V; Wierda, William G; Keating, Michael J; Shokat, Kevan M; Burger, Jan A

    2009-05-28

    Phosphoinositide 3-kinases (PI3Ks) are among the most frequently activated signaling pathways in cancer. In chronic lymphocytic leukemia (CLL), signals from the microenvironment are critical for expansion of the malignant B cells, and cause constitutive activation of PI3Ks. CXCR4 is a key receptor for CLL cell migration and adhesion to marrow stromal cells (MSCs). Because of the importance of CXCR4 and PI3Ks for CLL-microenvironment cross-talk, we investigated the activity of novel, isoform-selective PI3K inhibitors that target different isoforms of the p110-kDa subunit. Inhibition with p110alpha inhibitors (PIK-90 and PI-103) resulted in a significant reduction of chemotaxis and actin polymerization to CXCL12 and reduced migration beneath MSC (pseudoemperipolesis). Western blot and reverse phase protein array analyses consistently demonstrated that PIK-90 and PI-103 inhibited phosphorylation of Akt and S6, whereas p110delta or p110beta/p110delta inhibitors were less effective. In suspension and MSC cocultures, PI-103 and PIK-90 were potent inducers of CLL cell apoptosis. Moreover, these p110alpha inhibitors enhanced the cytotoxicity of fludarabine and reversed the protective effect of MSC on fludarabine-induced apoptosis. Collectively, our data demonstrate that p110alpha inhibitors antagonize stromal cell-derived migration, survival, and drug-resistance signals and therefore provide a rational to explore the therapeutic activity of these promising agents in CLL.

  8. The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma

    SciTech Connect

    Baumann, Philipp Mandl-Weber, Sonja; Oduncu, Fuat; Schmidmaier, Ralf

    2009-02-01

    NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

  9. Extended treatment with selective phosphatidylinositol 3-kinase and mTOR inhibitors has effects on metabolism, growth, behaviour and bone strength.

    PubMed

    Smith, Greg C; Ong, Wee-Kiat; Costa, Jessica L; Watson, Maureen; Cornish, Jillian; Grey, Andrew; Gamble, Greg D; Dickinson, Michelle; Leung, Sophie; Rewcastle, Gordon W; Han, Weiping; Shepherd, Peter R

    2013-11-01

    The class I phosphatidylinositol 3-kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small-molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on-target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small-molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro-CT and three-point bending, respectively. Surprisingly, after sustained dosing with pan-PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug-naïve animals treated with the same drugs. However, pan-PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health.

  10. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

    PubMed

    Aragoneses-Fenoll, L; Montes-Casado, M; Ojeda, G; Acosta, Y Y; Herranz, J; Martínez, S; Blanco-Aparicio, C; Criado, G; Pastor, J; Dianzani, U; Portolés, P; Rojo, J M

    2016-04-15

    Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

  11. Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

    SciTech Connect

    Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.

    2008-01-01

    Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K?/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

  12. VEGF secretion by neuroendocrine tumor cells is inhibited by octreotide and by inhibitors of the PI3K/AKT/mTOR pathway.

    PubMed

    Villaume, Karine; Blanc, Martine; Gouysse, Géraldine; Walter, Thomas; Couderc, Christophe; Nejjari, Mimoun; Vercherat, Cécile; Cordier-Bussat, Martine; Roche, Colette; Scoazec, Jean-Yves

    2010-01-01

    Gastroenteropancreatic (GEP) endocrine tumors are hypervascular tumors able to synthesize and secrete high amounts of VEGF. We aimed to study the regulation of VEGF production in GEP endocrine tumors and to test whether some of the drugs currently used in their treatment, such as somatostatin analogues and mTOR inhibitors, may interfere with VEGF secretion. We therefore analyzed the effects of the somatostatin analogue octreotide, the mTOR inhibitor rapamycin, the PI3K inhibitor LY294002, the MEK1 inhibitor PD98059 and the p38 inhibitor SB203850 on VEGF secretion, assessed by ELISA and Western blotting, in three murine endocrine cell lines, STC-1, INS-r3 and INS-r9. Octreotide and rapamycin induced a significant decrease in VEGF production by all three cell lines; LY294002 significantly inhibited VEGF production by STC-1 and INS-r3 only. We detected no effect of PD98059 whereas SB203850 significantly inhibited VEGF secretion in INS-r3 and INS-r9 cells only. By Western blotting analysis, we observed decreased intracellular levels of VEGF and HIF-1alpha under octreotide, rapamycin and LY294002. For rapamycin and LY294002, this effect was likely mediated by the inhibition of the mTOR/HIF-1/VEGF pathway. In addition to its well-known anti-secretory effects, octreotide may also act through the inhibition of the PI3K/Akt pathway, as suggested by the decrease in Akt phosphorylation detected in all three cell lines. In conclusion, our study points out to the complex regulation of VEGF synthesis and secretion in neoplastic GEP endocrine cells and suggests that the inhibition of VEGF production by octreotide and rapamycin may contribute to their therapeutic effects.

  13. FLT3 kinase inhibitor TTT-3002 overcomes both activating and drug resistance mutations in FLT3 in acute myeloid leukemia

    PubMed Central

    Ma, Hayley S.; Nguyen, Bao; Duffield, Amy S.; Li, Li; Galanis, Allison; Williams, Allen B.; Brown, Patrick A.; Levis, Mark J.; Leahy, Daniel J.; Small, Donald

    2014-01-01

    There have been a number of clinical trials testing the efficacy of FLT3 tyrosine kinase inhibitors (TKIs) in acute myeloid leukemia (AML). patients harboring a constitutively activating mutation in FLT3 However, there has been limited efficacy, most often due to inadequate achievement of FLT3 inhibition through a variety of mechanisms In a previous study, TTT-3002 was identified as a novel FLT3 inhibitor with the most potent activity to date against FLT3 internal tandem duplication (FLT3/ITD) mutations Here the activity of TTT-3002 is demonstrated against a broad spectrum of FLT3 activating point mutations (FLT3/PMs), including the most frequently occurring D835 mutations The compound is also active against a number of point mutations selected for in FLT3/ITD alleles that confer resistance to other TKIs, including the F691L gatekeeper mutation TTT-3002 maintains activity against relapsed AML patient samples that are resistant to sorafenib and AC220 Studies utilizing human plasma samples from healthy donors and AML patients indicate that TTT-3002 is only moderately protein bound compared to several other TKIs currently in clinical trials Tumor burden of mice in a FLT3 TKI-resistant transplant model is significantly improved by oral dosing of TTT-3002 Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKIs in the treatment of FLT3-mutant AML PMID:25060518

  14. The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase alpha leading to phosphocholine and total choline decrease detected by magnetic resonance spectroscopy.

    PubMed

    Al-Saffar, Nada M S; Jackson, L Elizabeth; Raynaud, Florence I; Clarke, Paul A; Ramírez de Molina, Ana; Lacal, Juan C; Workman, Paul; Leach, Martin O

    2010-07-01

    The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

  15. Phosphatidylinositol 3-kinaseinhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals

    PubMed Central

    Herman, Sarah E. M.; Gordon, Amber L.; Wagner, Amy J.; Heerema, Nyla A.; Zhao, Weiqiang; Flynn, Joseph M.; Jones, Jeffrey; Andritsos, Leslie; Puri, Kamal D.; Lannutti, Brian J.; Giese, Neill A.; Zhang, Xiaoli; Wei, Lai; Byrd, John C.

    2010-01-01

    Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101–mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell–activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders. PMID:20522708

  16. G-749, a novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of acute myeloid leukemia

    PubMed Central

    Lee, Hee Kyu; Kim, Hong Woo; Lee, In Yong; Lee, Jungmi; Lee, Jaekyoo; Jung, Dong Sik; Lee, Sang Yeop; Park, Sung Ho; Hwang, Haejun; Choi, Jang-Sik; Kim, Jung-Ho; Kim, Se Won; Kim, Jung Keun; Cools, Jan; Koh, Jong Sung

    2014-01-01

    Aberrant activations of Fms-like tyrosine receptor kinase (FLT) 3 are implicated in the pathogenesis of 20% to 30% of patients with acute myeloid leukemia (AML). G-749 is a novel FLT3 inhibitor that showed potent and sustained inhibition of the FLT3 wild type and mutants including FLT3-ITD, FLT3-D835Y, FLT3-ITD/N676D, and FLT3-ITD/F691L in cellular assays. G-749 retained its inhibitory potency in various drug-resistance milieus such as patient plasma, FLT3 ligand surge, and stromal protection. Furthermore, it displayed potent antileukemic activity in bone marrow blasts from AML patients regardless of FLT3 mutation status, including those with little or only minor responses to AC220 or PKC412. Oral administration of G-749 yielded complete tumor regression and increased life span in animal models. Thus, G-749 appears to be a promising next-generation drug candidate for the treatment of relapsed and refractory AML patients with various FLT3-ITD/FLT3-TKD mutants and further shows the ability to overcome drug resistance. PMID:24532805

  17. HS-133, a novel fluorescent phosphatidylinositol 3-kinase inhibitor as a potential imaging and anticancer agent for targeted therapy

    PubMed Central

    Lee, Hyunseung; Son, Mi Kwon; Yun, Sun-Mi; Ahn, Sung-Hoon; Lee, Kyeong-Ryoon; Lee, Soyoung; Kim, Donghee; Hong, Sungwoo; Hong, Soon-Sun

    2014-01-01

    As PI3K/Akt signaling is frequently deregulated in a wide variety of human tumors, PI3K inhibitors are an emerging class of drugs for cancer treatment. The monitoring of the drug behavior and distribution in the biological system can play an important role for targeted therapy and provide information regarding the response or resistance to available therapies. In this study, therefore, we have developed a family of xanthine derivatives, serving as a dual function exhibiting fluorescence, as well as inhibiting PI3K. Among them, HS-133 showed anti-proliferative effects and was monitored for its subcellular localization by a fluorescence microscopy. HS-133 suppressed the PI3K/Akt pathway and induced cell cycle arrest at the G0/G1 phase. The induction of apoptosis by HS-133 was confirmed by the increases of the cleaved PARP, caspase-3, and caspase-8. Furthermore, HS-133 decreased the protein expression of HIF-1α and VEGF, as well inhibited the tube formation and migration of the human umbilical vein endothelial cells. In vivo imaging also showed that tumors were visualized fluorescent with HS-133, and its oral administration significantly inhibited the growth of tumor in SkBr3 mouse xenograft models. Thus, we suggest that HS-133 may be used as a fluorescent anticancer agent against human breast cancer. PMID:25338206

  18. Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick

    PubMed Central

    Penha, Alexandra Marcha; Burkhardt, Eva; Schaeffel, Frank

    2012-01-01

    Purpose Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses. Methods Chicks were treated with either +7 D, −7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-AktThr308, and anti-phospho-Erk1/2(Thr202/Tyr204) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated. Results Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens–treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor

  19. Selective inhibitors of phosphoinositide 3-kinase delta: modulators of B-cell function with potential for treating autoimmune inflammatory diseases and B-cell malignancies

    PubMed Central

    Puri, Kamal D.; Gold, Michael R.

    2012-01-01

    The delta isoform of the p110 catalytic subunit (p110δ) of phosphoinositide 3-kinase is expressed primarily in hematopoietic cells and plays an essential role in B-cell development and function. Studies employing mice lacking a functional p110δ protein, as well as the use of highly-selective chemical inhibitors of p110δ, have revealed that signaling via p110δ-containing PI3K complexes (PI3Kδ) is critical for B-cell survival, migration, and activation, functioning downstream of key receptors on B cells including the B-cell antigen receptor, chemokine receptors, pro-survival receptors such as BAFF-R and the IL-4 receptor, and co-stimulatory receptors such as CD40 and Toll-like receptors (TLRs). Similarly, this PI3K isoform plays a key role in the survival, proliferation, and dissemination of B-cell lymphomas. Herein we summarize studies showing that these processes can be inhibited in vitro and in vivo by small molecule inhibitors of p110δ enzymatic activity, and that these p110δ inhibitors have shown efficacy in clinical trials for the treatment of several types of B-cell malignancies including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PI3Kδ also plays a critical role in the activation, proliferation, and tissue homing of self-reactive B cells that contribute to autoimmune diseases, in particular innate-like B-cell populations such as marginal zone (MZ) B cells and B-1 cells that have been strongly linked to autoimmunity. We discuss the potential utility of p110δ inhibitors, either alone or in combination with B-cell depletion, for treating autoimmune diseases such as lupus, rheumatoid arthritis, and type 1 diabetes. Because PI3Kδ plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, PI3Kδ inhibitors may represent a promising therapeutic approach for treating these diseases. PMID:22936933

  20. The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells.

    PubMed

    Fiorcari, Stefania; Brown, Wells S; McIntyre, Bradley W; Estrov, Zeev; Maffei, Rossana; O'Brien, Susan; Sivina, Mariela; Hoellenriegel, Julia; Wierda, William G; Keating, Michael J; Ding, Wei; Kay, Neil E; Lannutti, Brian J; Marasca, Roberto; Burger, Jan A

    2013-01-01

    CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

  1. The PI3-Kinase Delta Inhibitor Idelalisib (GS-1101) Targets Integrin-Mediated Adhesion of Chronic Lymphocytic Leukemia (CLL) Cell to Endothelial and Marrow Stromal Cells

    PubMed Central

    Fiorcari, Stefania; Brown, Wells S.; McIntyre, Bradley W.; Estrov, Zeev; Maffei, Rossana; O’Brien, Susan; Sivina, Mariela; Hoellenriegel, Julia; Wierda, William G.; Keating, Michael J.; Ding, Wei; Kay, Neil E.; Lannutti, Brian J.; Marasca, Roberto; Burger, Jan A.

    2013-01-01

    CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood. PMID:24376763

  2. Enhanced Efficacy of 5-Fluorouracil in Combination with a Dual Histone Deacetylase and Phosphatidylinositide 3-Kinase Inhibitor (CUDC-907) in Colorectal Cancer Cells

    PubMed Central

    Hamam, Rimi; Ali, Dalia; Vishnubalaji, Radhakrishnan; Alsaaran, Zaid F.; Chalisserry, Elna Paul; Alfayez, Musaad; Aldahmash, Abdullah; Alajez, Nehad M.

    2017-01-01

    Background/Aims: 5-Fluorouracil (5-FU) is widely used in the treatment of patients with colorectal cancer (CRC). However, the efficacy of 5-FU as a single agent is limited, with multiple undesired side effects. Therefore, the aim of the current study was to assess the efficacy of CUDC-907 (a dual inhibitor of histone deacetylase and phosphatidylinositide 3-kinase) in combination with 5-FU against CRC cells. Materials and Methods: Cell viability was determined using AlamarBlue and colony formation assays. Acridine orange/ethidium bromide staining and flow cytometry were used to measure apoptotic and necrotic events, as well as cell cycle progression. Immunoblotting was used to assess acetylation of histone H3 and phosphorylation of AKT. Results: Our data revealed enhanced toxicity of CUDC-907 against HCT116, RKO, COLO-205, and HT-29 CRC cells when combined with 5-FU. Similarly, the colony formation capability of HCT116 cells was suppressed by the combination treatment. Cells treated with CUDC-907 and 5-FU underwent apoptosis and necrosis, and exhibited increased polyploidy. Furthermore, CRC cells treated with CUDC-907 exhibited a higher degree of histone H3 lysine 9 acetylation (H3K9ac) and reduced AKT phosphorylation (Ser473). Conclusion: Our data revealed, for the first time, the enhanced inhibitory effect of CUDC-907 against CRC cells when combined with 5-FU, supporting the application of this combination as a potential therapeutic strategy in CRC treatment. PMID:28139498

  3. The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways.

    PubMed

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T; Portell, Craig A; Lannutti, Brian J; Almasan, Alexandru; Hsi, Eric D

    2013-10-01

    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

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

    PubMed

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

    2002-02-07

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

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

    PubMed

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

    2010-04-01

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

  6. Potential of the Akt inhibitor LY294005 to antagonize the efficacy of Cisplatin against HCT116 tumor cells in a DNA mismatch repair-dependent manner.

    PubMed

    Fedier, Andre; Erdmann, Ruediger; Boulikas, Teni; Fink, Daniel

    2006-11-01

    Human colorectal adenocarcinoma sublines either deficient (HCT116+ch2) or proficient (HCT116+ch3) in the function of MLH1, one of five proteins crucial to DNA mismatch repair (MMR), were used to investigate whether the Akt-specific inhibitor LY294005 could not only increase the efficacy of platinum drugs in HCT116 cells in general but also increase the efficacy of the cisplatinum compounds Cisplatin and Lipoplatin specifically in MLH1-deficient, Cisplatin- and Lipoplatin-resistant HCT116 cells. We report that, under the conditions it increased the efficacy of Docetaxel and did not affect that of 6-thioguanine, LY294005 decreased the sensitivity of both sublines to Cisplatin, Lipoplatin, Oxaliplatin, and Lipoxal. Notably, the LY294005-imposed decrease was significantly higher in the MLH1-proficient than in the MLH1-deficient subline with Cisplatin and Lipoplatin, whereas it was nearly the same in both sublines with Oxaliplatin and Lipoxal. These LY294005-imposed changes in drug sensitivity, i.e. increase with Docetaxel and decreases with platinum compounds, were not associated with the concomitant abrogation in the levels of phospho-Aktser473. Analogous changes in drug sensitivity were also observed with the PI3-kinase inhibitor LY294002, but these changes were associated with complete abrogation of phospho-Aktser473. These observations suggest a possible relationship between MMR-mediated cisplatinum DNA damage signaling and the Akt signaling pathway, e.g. a common target for both pathways. A possibly novel property of Akt in aggravating drug sensitivity may also be proposed.

  7. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway.

    PubMed

    Chen, Jiun-Han; Hsiao, George; Lee, An-Rong; Wu, Chin-Chen; Yen, Mao-Hsiung

    2004-04-01

    Andrographolide (Andro), an active component isolated from the Chinese official herbal Andrographis paniculata, which has been reported to prevent oxygen radical production and thus prevent inflammatory diseases. In this study, we investigated the molecular mechanisms and signaling pathways by which Andro protects human umbilical vein endothelial cells (HUVECs) from growth factor (GF) deprivation-induced apoptosis. Results demonstrated that HUVECs undergo apoptosis after 18 hr of GF deprivation but that this cell death was suppressed by the addition of Andro in a concentration-dependent manner (1-100 microM). Andro suppresses the mitochondrial pathway of apoptosis by inhibiting release of cytochrome c into the cytoplasm and dissipation of mitochondrial potential (Deltapsi(m)), as a consequence, prevented caspase-3 and -9 activation. Treatment of endothelial cells with Andro-induced activation of the protein kinase Akt, an anti-apoptotic signal, and phosphorylation of BAD, a down-stream target of Akt. Suppression of Akt activity by wortmannin, by LY-294002 and by using a dominant negative Akt mutant abolished the anti-apoptotic effect of Andro. In contrast, the ERK1/2 activities were not affected by Andro. The ERK1/2 inhibitor, PD98059 failed to antagonize the protective effect of Andro. In conclusion, Andro exerts its anti-apoptotic potential via activation of the Akt-BAD pathway in HUVECs and thus may represent a candidate of therapeutic agent for atherosclerosis.

  8. Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase.

    PubMed

    Tornieri, Karine; Welshhans, Kristy; Geddis, Matthew S; Rehder, Vincent

    2006-04-01

    Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.

  9. PI3K Inhibition Augments the Therapeutic Efficacy of a 3a-aza-Cyclopenta[α]indene Derivative in Lung Cancer Cells12

    PubMed Central

    Sanjiv, Kumar; Chen, Chi-Wei; Kakadiya, Rajesh; Tala, Satishkumar; Suman, Sharda; Wu, Ming-Hsi; Chen, Yen-Hui; Su, Tsann-Long; Lee, Te-Chang

    2014-01-01

    The synergistic targeting of DNA damage and DNA repair is a promising strategy for the development of new chemotherapeutic agents for human lung cancer. The DNA interstrand cross-linking agent BO-1509, a derivative of 3a-aza-cyclopenta[α]indene, was synthesized and combined with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 to treat human lung cancer cells. Our results showed that the BO-1509 and LY294002 combination synergistically killed lung cancer cells in culture and also suppressed the growth of lung cancer xenografts in mice, including those derived from gefitinib-resistant cells. We also found that LY294002 suppressed the induction of several DNA repair proteins by BO-1509 and inhibited the nuclear translocation of Rad51. On the basis of the results of the γH2AX foci formation assays, LY294002 apparently inhibited the repair of DNA damage that was induced by BO-1509. According to the complete blood profile, biochemical enzyme analysis, and histopathologic analysis of major organs, no apparent toxicity was observed in mice treated with BO-1509 alone or in combination with LY294002. Our results suggest that the combination of a DNA cross-linking agent with a PI3K inhibitor is a feasible strategy for the treatment of patients with lung cancer. PMID:24913674

  10. Autophagy Inhibitors as a Potential Antiamoebic Treatment for Acanthamoeba Keratitis

    PubMed Central

    Moon, Eun-Kyung; Kim, So-Hee; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung

    2015-01-01

    Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis. PMID:25896709

  11. Autophagy inhibitors as a potential antiamoebic treatment for Acanthamoeba keratitis.

    PubMed

    Moon, Eun-Kyung; Kim, So-Hee; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung; Kong, Hyun-Hee

    2015-07-01

    Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis.

  12. Resveratrol inhibits TNF-α-induced IL-1β, MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of PI3kinase/Akt pathway.

    PubMed

    Tian, Jing; Chen, Jin-wei; Gao, Jie-sheng; Li, Len; Xie, Xi

    2013-07-01

    Resveratrol (trans-3,4'-trihydroxystilbene), a natural phytoalexin, possesses anti-inflammatory, anti-proliferative, and immunomodulatory properties and has the potential for treating inflammatory disorders. The present study was designed to investigate the effects of resveratrol on TNF-α-induced inflammatory cytokines production of IL-1β and MMP3 in Rheumatoid arthritis (RA) Fibroblast-like synoviocytes (FLS) and further to explore the role of PI3K/Akt signaling pathway by which resveratrol modulates those cytokines production. The levels of IL-1β, MMP-3 in cultural supernatants among groups were measured by enzyme-linked immunosorbent assay. Messenger RNA expression of IL-1β and MMP-3 in RA FLS was analyzed using a reverse transcription-polymerase chain reaction. Western blot analysis was used to detect proteins expression in RA FLS intervened by resveratrol. Resveratrol inhibited both mRNA and proteins expressions of IL-1β and MMP-3 on RA FLS in a dose-dependent manner. Resveratrol also decreased significantly the expression of phosphorylated Akt dose dependently. Activation of PI3K/Akt signaling pathway exists in TNF-α-induced production of IL-1β and MMP3 on RA FLS, which is hampered by PI3K inhibitor LY294002. Immunofluorescence staining showed that TNF-α alone increased the production of P-Akt, whereas LY294002 and 50 μM resveratrol suppressed the TNF-α-stimulated expression of P-Akt. Resveratrol attenuates TNF-α-induced production of IL-1β and MMP-3 via inhibition of PI3K-Akt signaling pathway in RA FLS, suggesting that resveratrol plays an anti-inflammatory role and might have beneficial effects in preventing and treating RA.

  13. Factors Influencing the Central Nervous System Distribution of a Novel Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GSK2126458: Implications for Overcoming Resistance with Combination Therapy for Melanoma Brain Metastases

    PubMed Central

    Vaidhyanathan, Shruthi; Wilken-Resman, Brynna; Ma, Daniel J.; Parrish, Karen E.; Mittapalli, Rajendar K.; Carlson, Brett L.; Sarkaria, Jann N.

    2016-01-01

    Small molecule inhibitors targeting the mitogen-activated protein kinase pathway (Braf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) have had success in extending survival for patients with metastatic melanoma. Unfortunately, resistance may occur via cross-activation of alternate signaling pathways. One approach to overcome resistance is to simultaneously target the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway. Recent reports have shown that GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl) benzenesulfonamide], a dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor, can overcome acquired resistance to Braf and mitogen-activated protein kinase kinase inhibitors in vitro. These resistance mechanisms may be especially important in melanoma brain metastases because of limited drug delivery across the blood–brain barrier. The purpose of this study was to investigate factors that influence the brain distribution of GSK2126458 and to examine the efficacy of GSK2126458 in a novel patient-derived melanoma xenograft (PDX) model. Both in vitro and in vivo studies indicate that GSK2126458 is a substrate for P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), two dominant active efflux transporters in the blood–brain barrier. The steady-state brain distribution of GSK2126458 was 8-fold higher in the P-gp/Bcrp knockout mice compared with the wild type. We also observed that when simultaneously infused to steady state, GSK212658, dabrafenib, and trametinib, a rational combination to overcome mitogen-activated protein kinase inhibitor resistance, all had limited brain distribution. Coadministration of elacridar, a P-gp/Bcrp inhibitor, increased the brain distribution of GSK2126458 by approximately 7-fold in wild-type mice. In the PDX model, GSK2126458 showed efficacy in flank tumors but was ineffective in intracranial melanoma. These results show

  14. Dual inhibition of histone deacetylases and phosphoinositide 3-kinases: effects on Burkitt lymphoma cell growth and migration.

    PubMed

    Ferreira, Ana Carolina dos Santos; de-Freitas-Junior, Julio Cesar Madureira; Morgado-Díaz, Jose Andres; Ridley, Anne J; Klumb, Claudete Esteves

    2016-04-01

    Burkitt lymphoma is a highly aggressive non-Hodgkin lymphoma that is characterized by MYC deregulation. Recently, the PI3K pathway has emerged as a cooperative prosurvival mechanism in Burkitt lymphoma. Despite the highly successful results of treatment that use high-dose chemotherapy regimens in pediatric Burkitt lymphoma patients, the survival rate of pediatric patients with progressive or recurrent disease is low. PI3Ks are also known to regulate cell migration, and abnormal cell migration may contribute to cancer progression and dissemination in Burkitt lymphoma. Little is known about Burkitt lymphoma cell migration, but the cooperation between MYC and PI3K in Burkitt lymphoma pathogenesis suggests that a drug combination could be used to target the different steps involved in Burkitt lymphoma cell dissemination and disease progression. The aim of this study was to investigate the effects of the histone deacetylase inhibitor suberoylanilide hydroxamic acid combined with the PI3K inhibitor LY294002 on Burkitt lymphoma cell growth and migration. The combination enhanced the cell growth inhibition and cell-cycle arrest induced by the PI3K inhibitor or histone deacetylase inhibitor individually. Moreover, histone deacetylase inhibitor/PI3K inhibitor cotreatment suppressed Burkitt lymphoma cell migration and decreased cell polarization, Akt and ERK1/2 phosphorylation, and leads to RhoB induction. In summary, the histone deacetylase inhibitor/PI3Ki combination inhibits cell proliferation and migration via alterations in PI3K signaling and histone deacetylase activity, which is involved in the acetylation of α-tubulin and the regulation of RhoB expression.

  15. Cross-species Proteomics Reveals Specific Modulation of Signaling in Cancer and Stromal Cells by Phosphoinositide 3-kinase (PI3K) Inhibitors*

    PubMed Central

    Rajeeve, Vinothini; Vendrell, Iolanda; Wilkes, Edmund; Torbett, Neil; Cutillas, Pedro R.

    2014-01-01

    The tumor microenvironment plays key roles in cancer biology, but its impact on the regulation of signaling pathway activity in cancer cells has not been systemically investigated. We designed an analytical strategy that allows differential analysis of signaling between cancer and stromal cells present in tumor xenografts. We used this approach to investigate how in vivo growth conditions and PI3K inhibitors regulate pathway activities in both cancer and stromal cell populations. We found that, despite inducing more modest changes in protein expression, in vivo growing conditions extensively rewired protein kinase networks in cancer cells. As a result, different sets of phosphorylation sites were modulated by PI3K inhibitors in cancer cells growing in tumors relative to when these cells were in culture. The p110δ PI3K-selective compound CAL-101 (Idelalisib) did not inhibit markers of PI3K activity in cancer or stromal cells; however, unexpectedly, it induced phosphorylation on SQ motifs in both subpopulations of tumor cells in vivo but not in vitro. Thus, the interaction between cancer cells and the stroma modulated the ability of PI3K inhibitors to induce the activation of apoptosis in solid tumors. Our study provides proof-of-principle of a proteomics workflow for measuring signaling specifically in cancer and stromal cells and for investigating how cancer biochemistry is modulated in vivo. PMID:24648465

  16. The pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia

    PubMed Central

    Allegretti, Matteo; Ricciardi, Maria Rosaria; Licchetta, Roberto; Mirabilii, Simone; Orecchioni, Stefania; Reggiani, Francesca; Talarico, Giovanna; Foà, Roberto; Bertolini, Francesco; Amadori, Sergio; Torrisi, Maria Rosaria; Tafuri, Agostino

    2015-01-01

    Aberrant activation of the PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represent a potential therapeutic approach in AML. The aim of this study was to evaluate the pre-clinical activity of NVP-BKM120 (BKM120), a selective pan-class I PI3K inhibitor, on AML cell lines and primary samples. Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest and significant apoptosis in a dose- and time-dependent manner in AML cells but not in the normal counterpart. BKM120-induced cytotoxicity is associated with a profound modulation of metabolic behaviour in both cell lines and primary samples. In addition, BKM120 synergizes with the glycolitic inhibitor dichloroacetate enhancing apoptosis induction at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibited leukemia progression and improved the overall survival of treated mice. Taken together, our findings indicate that BKM120, alone or in combination with other drugs, has a significant anti-leukemic activity supporting its clinical development as a novel therapeutic agent in AML. PMID:26674543

  17. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin.

    PubMed

    Singleton, Patrick A; Dudek, Steven M; Chiang, Eddie T; Garcia, Joe G N

    2005-10-01

    Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 microM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase alpha and beta catalytic subunits, the Rac1 GEF, Tiam1, and alpha-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, alpha-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and alpha-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both alpha-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for alpha-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.

  18. Phase I Study of Apitolisib (GDC-0980), Dual Phosphatidylinositol-3-Kinase and Mammalian Target of Rapamycin Kinase Inhibitor, in Patients with Advanced Solid Tumors

    PubMed Central

    Dolly, Saoirse O.; Wagner, Andrew J.; Bendell, Johanna C.; Kindler, Hedy L.; Krug, Lee M.; Seiwert, Tanguy Y.; Zauderer, Marjorie G.; Lolkema, Martijn P.; Apt, Doris; Yeh, Ru-Fang; Fredrickson, Jill O.; Spoerke, Jill M.; Koeppen, Hartmut; Ware, Joseph A.; Lauchle, Jennifer O.; Burris, Howard A.; de Bono, Johann S.

    2016-01-01

    Purpose This first-in-human phase I trial assessed the safety, tolerability, and preliminary anti-tumor activity of apitolisib (GDC-0980), a dual inhibitor of class I phosphatidylinositol-3-(PI3K) and mammalian target of rapamycin (mTOR) kinases. Experimental Design Once-daily (QD) oral apitolisib was administered to patients with solid tumors for days 1-21 or 1-28 of 28-day cycles. Pharmacokinetic and pharmacodynamic parameters were assessed. Results Overall, 120 patients were treated at doses between 2-70 mg. The commonest ≥G3 toxicities related to apitolisib at the recommended phase 2 dose (RP2D) at 40mg QD included hyperglycemia (18%), rash (14%), liver dysfunction (12%), diarrhea (10%), pneumonitis (8%), mucosal inflammation (6%), and fatigue (4%). Dose-limiting toxicities (one patient each) were G4 fasting hyperglycemia at 40 mg (21/28-schedule), and G3 maculopapular rash and G3 fasting hyperglycemia at 70 mg (21/28-schedule). The pharmacokinetic profile was dose-proportional. Phosphorylated serine-473 AKT levels were suppressed by ≥90% in platelet-rich plasma within 4 hours at the maximum tolerated dose (50 mg). Pharmacodynamic decreases in FDG-PET uptake of >25% occurred in 66% (21/32) of patients dosed at 40 mg QD. Evidence of single agent activity included ten RECIST partial responses (confirmed for peritoneal mesothelioma, PIK3CA mutant head- and-neck cancer, and three pleural mesotheliomas). Conclusion Apitolisib exhibited dose-proportional pharmacokinetics with target modulation at doses ≥16 mg. The RP2D was 40 mg QD 28/28-schedule; severe on-target toxicities were apparent at ≥40 mg, particularly pneumonitis. Apitolisib was reasonably tolerated at 30 mg, the selected dose for pleural mesothelioma patients given limited respiratory reserve. Modest but durable anti-tumor activity was demonstrated. PMID:26787751

  19. HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage.

    PubMed

    Pääjärvi, Gerd; Roudier, Emilie; Crisby, Milita; Högberg, Johan; Stenius, Ulla

    2005-03-01

    3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are widely used cholesterol-lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53-specific ubiquitin ligase. It was found that pravastatin induced Mdm2 phosphorylation at Ser166 and at 2A10 antibody-specific epitopes in HepG2 cells, while mRNA levels were unchanged. Furthermore, pravastatin was found to induce phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated by an inhibitor of mTOR, rapamycin, but not by the PI3-kinase inhibitors LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to active Mdm2 and has been shown to increase its ubiquitin ligase activity and lead to increased p53 degradation. Our data show that statins attenuated the p53 response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin pretreatment attenuated the p53 response to DNA damage induced by 5-fluorouracil and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the DNA-damaged cells, half-lives of Mdm2 and p53 were decreased by statins, indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53 degradation. The induction of p53 responsive genes and apoptosis was attenuated. Mdm2 and p53 were also studied in vivo in rat liver employing immunohistochemistry, and it was found that constitutive Mdm2 expression was changed in livers of pravastatin-treated rats. We also show that the p53 response to a challenging dose of diethylnitrosamine was attenuated in hepatocytes in situ and in primary cultures of hepatocytes by pravastatin pretreatment. Taken together, these data indicate that statins induce an mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes.

  20. Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

    PubMed

    Fujimoto, Masaru; Suda, Yasuyuki; Vernhettes, Samantha; Nakano, Akihiko; Ueda, Takashi

    2015-02-01

    The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.

  1. A Phase Ib Study of BEZ235, a Dual Inhibitor of Phosphatidylinositol 3-Kinase (PI3K) and Mammalian Target of Rapamycin (mTOR), in Patients With Advanced Renal Cell Carcinoma

    PubMed Central

    Molina, Ana M.; Lakhman, Yulia; Patil, Sujata; Woo, Kaitlin; DeLuca, John; Lee, Chung-Han; Hsieh, James J.; Feldman, Darren R.; Motzer, Robert J.; H. Voss, Martin

    2016-01-01

    Lessons Learned Our results highlight additional toxicities of dual PI3K/mTOR inhibition in the clinical setting that were unforeseen from preclinical models. Because of toxicity and lack of efficacy, BEZ235 should not be further developed in the current formulation for patients with renal cell carcinoma. Background. Allosteric inhibitors of the mammalian target of rapamycin complex 1 (mTORC1) are approved for advanced renal cell carcinoma (RCC). Preclinical models have suggested that dual inhibition of phosphatidylinositol 3-kinase (PI3K) and mTOR kinase may establish superior anticancer effect. We aimed to establish safety for BEZ235, a potent inhibitor of both PI3K and mTOR, in advanced RCC. Methods. Patients with advanced RCC who had previously failed standard therapy received escalating doses of BEZ235 in sachet formulation twice daily until progression or unacceptable toxicity. Primary endpoints were to identify the maximally tolerated dose (MTD) and to determine the recommended dose for the phase II study. Results. The study was terminated early because of high incidence of dose-limiting toxicities (DLTs) across all dose levels tested. Ten patients were treated with BEZ235—six with clear cell and four with non-clear cell subtypes. Five of these patients suffered DLTs: 2 of 2 patients in the original 400 mg b.i.d. cohort, 1 of 6 in the 200 mg b.i.d. cohort, and 2 of 2 in the 300 mg b.i.d. cohort. DLTs included fatigue, rash, nausea and vomiting, diarrhea, mucositis, anorexia, and dysgeusia. Five patients were evaluable for response: Two had stable disease as best response, and three had progressive disease. Conclusion. BEZ235 twice daily resulted in significant toxicity without objective responses; further development of this compound will not be pursued in this disease. PMID:27286790

  2. Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.

    PubMed

    Bago, Ruzica; Malik, Nazma; Munson, Michael J; Prescott, Alan R; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G; Alessi, Dario R

    2014-11-01

    The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the Ptd

  3. Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

    SciTech Connect

    Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi; Nawa, Takatoshi; Kodama, Takahiro; Shimizu, Satoshi; Hikita, Hayato; Hiramatsu, Naoki; Kanto, Tatsuya; Hayashi, Norio; Takehara, Tetsuo

    2011-08-19

    Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

  4. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    SciTech Connect

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-08-15

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  5. A randomized, phase 2 trial of docetaxel with or without PX-866, an irreversible oral phosphatidylinositol 3-kinase inhibitor, in patients with relapsed or metastatic head and neck squamous cell cancer

    PubMed Central

    Jimeno, Antonio; Bauman, Julie E.; Weissman, Charles; Adkins, Douglas; Schnadig, Ian; Beauregard, Patrice; Bowles, Daniel W.; Spira, Alexander; Levy, Benjamin; Seetharamu, Nagashree; Hausman, Diana; Walker, Luke; Rudin, Charles M.; Shirai, Keisuke

    2016-01-01

    SUMMARY Introduction The phosphotidylinositol-3 kinase (PI3K)/serine–threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is frequently altered in head and neck squamous cell cancer (HNSCC). PX-866 is an oral, irreversible, pan-isoform inhibitor of PI3K. Preclinical models revealed synergy with docetaxel and a phase 1 trial demonstrated tolerability of this combination. This randomized phase 2 study evaluated PX-866 combined with docetaxel in patients with advanced, refractory HNSCC. Methods Patients with locally advanced, recurrent or metastatic HNSCC who had received at least one and no more than two prior systemic treatment regimens were randomized (1:1) to a combination of docetaxel (75 mg/m2 IV every 21 days) with or without PX-866 (8 mg PO daily; Arms A and B, respectively). The primary endpoint was progression free survival (PFS). Secondary endpoints included objective response rate (RR), overall survival (OS), toxicity, and correlation of biomarker analyses with efficacy outcomes. Results 85 patients were enrolled. There was a non-significant improvement in response rate in the combination arm (14% vs. 5%; P = 0.13). Median PFS was 92 days in Arm A and 82 days in Arm B (P = 0.42). There was no difference in OS between the two arms (263 vs. 195 days; P = 0.62). Grade 3 or higher adverse events were infrequent, but more common in the combination arm with respect to diarrhea (17% vs. 2%), nausea (7% vs. 0%), and febrile neutropenia (21% vs. 5%); grade 3 or higher anemia was more frequent in arm B (7% vs. 27%). PIK3CA mutations or PTEN loss were infrequently observed. Conclusion The addition of PX-866 to docetaxel did not improve PFS, RR, or OS in patients with advanced, refractory HNSCC without molecular pre-selection. PMID:25593016

  6. First-in-human Phase I study of Pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors

    PubMed Central

    Baird, Richard; Kristeleit, Rebecca; Shah, Krunal; Moreno, Victor; Clarke, Paul A.; Raynaud, Florence I.; Levy, Gallia; Ware, Joseph A; Mazina, Kathryn; Lin, Ray; Wu, Jenny; Fredrickson, Jill; Spoerke, Jill M; Lackner, Mark R; Yan, Yibing; Friedman, Lori S.; Kaye, Stan B.; Derynck, Mika K.; Workman, Paul; de Bono, Johann S.

    2014-01-01

    Purpose This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal tolerated dose (MTD), dose limiting toxicities (DLTs), pharmacokinetics, pharmacodynamics and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent and selective inhibitor of the Class I phosphatidylinositol-3-kinases (PI3K). Patients and Methods Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450mg once-daily, initially on days 1-21 every 28 days and later, utilizing continuous dosing for selected dose levels. Pharmacodynamic studies incorporated 18F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma and tumor tissue. Results Pictilisib was well-tolerated. The most common toxicities were grade 1-2 nausea, rash and fatigue while the DLT was grade 3 maculopapular rash (450mg, 2 of 3 patients; 330mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in platelet rich plasma at 3 hours post-dose at the MTD and in tumor at pictilisib doses associated with AUC >20uM.hr. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. Conclusion Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100mg and signs of antitumor activity. The recommended Phase II dose was continuous dosing at 330mg once-daily. PMID:25370471

  7. Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway.

    PubMed

    Torroglosa, Ana; Murillo-Carretero, Maribel; Romero-Grimaldi, Carmen; Matarredona, Esperanza R; Campos-Caro, Antonio; Estrada, Carmen

    2007-01-01

    Nitric oxide (NO) inhibits proliferation of subventricular zone (SVZ) neural precursor cells in adult mice in vivo under physiological conditions. The mechanisms underlying this NO effect have now been investigated using SVZ-derived neural stem cells, which generate neurospheres in vitro when stimulated by epidermal growth factor (EGF). In these cultures, NO donors decreased the number of newly formed neurospheres as well as their size, which indicates that NO was acting on the neurosphere-forming neural stem cells and the daughter neural progenitors. The effect of NO was cytostatic, not proapoptotic, and did not involve cGMP synthesis. Neurosphere cells expressed the neuronal and endothelial isoforms of NO synthase (NOS) and produced NO in culture. Inhibition of NOS activity by N(omega)-nitro-L-arginine methylester (L-NAME) promoted neurosphere formation and growth, thus revealing an autocrine/paracrine action of NO on the neural precursor cells. Both exogenous and endogenous NO impaired the EGF-induced activation of the EGF receptor (EGFR) tyrosine kinase and prevented the EGF-induced Akt phosphorylation in neurosphere cells. Inhibition of the phosphoinositide-3-kinase (PI3-K)/Akt pathway by LY294002 significantly reduced the number of newly formed neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal. Chronic administration of l-NAME to adult mice enhanced phospho-Akt staining in the SVZ and reduced nuclear p27(Kip1) in the SVZ and olfactory bulb. The inhibition of EGFR and PI3-K pathway by NO explains, at least in part, its antimitotic effect on neurosphere cells and may be a mechanism involved in the physiological role of NO as a negative regulator of SVZ neurogenesis in adult mice.

  8. The phosphoinositide 3-kinase pathway.

    PubMed

    Cantley, Lewis C

    2002-05-31

    Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

  9. First-in-human phase I study of copanlisib (BAY 80-6946), an intravenous pan-class I phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors and non-Hodgkin's lymphomas

    PubMed Central

    Patnaik, A.; Appleman, L. J.; Tolcher, A. W.; Papadopoulos, K. P.; Beeram, M.; Rasco, D. W.; Weiss, G. J.; Sachdev, J. C.; Chadha, M.; Fulk, M.; Ejadi, S.; Mountz, J. M.; Lotze, M. T.; Toledo, F. G. S.; Chu, E.; Jeffers, M.; Peña, C.; Xia, C.; Reif, S.; Genvresse, I.; Ramanathan, R. K.

    2016-01-01

    Background To evaluate the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of copanlisib, a phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors or non-Hodgkin's lymphoma (NHL). Patients and methods Phase I dose-escalation study including patients with advanced solid tumors or NHL, and a cohort of patients with type 2 diabetes mellitus. Patients received three weekly intravenous infusions of copanlisib per 28-day cycle over the dose range 0.1–1.2 mg/kg. Plasma copanlisib levels were analyzed for pharmacokinetics. Biomarker analysis included PIK3CA, KRAS, BRAF, and PTEN mutational status and PTEN immunohistochemistry. Whole-body [18F]-fluorodeoxyglucose positron emission tomography (18FDG-PET) was carried out at baseline and following the first dose to assess early pharmacodynamic effects. Plasma glucose and insulin levels were evaluated serially. Results Fifty-seven patients received treatment. The MTD was 0.8 mg/kg copanlisib. The most frequent treatment-related adverse events were nausea and transient hyperglycemia. Copanlisib exposure was dose-proportional with no accumulation; peak exposure positively correlated with transient hyperglycemia post-infusion. Sixteen of 20 patients treated at the MTD had reduced 18FDG-PET uptake; 7 (33%) had a reduction >25%. One patient achieved a complete response (CR; endometrial carcinoma exhibiting both PIK3CA and PTEN mutations and complete PTEN loss) and two had a partial response (PR; both metastatic breast cancer). Among the nine NHL patients, all six with follicular lymphoma (FL) responded (one CR and five PRs) and one patient with diffuse large B-cell lymphoma had a PR by investigator assessment; two patients with FL who achieved CR (per post hoc independent radiologic review) were on treatment >3 years. Conclusion Copanlisib, dosed intermittently on days 1, 8, and 15 of a 28-day cycle, was well tolerated and the MTD was determined to be 0.8 mg/kg. Copanlisib

  10. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

    PubMed Central

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2010-01-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton. PMID:16204406

  11. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

    PubMed

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2006-04-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

  12. Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell lines.

    PubMed

    Mei, Chuanzhong; Sun, Lidong; Liu, Yonglei; Yang, Yong; Cai, Xiumei; Liu, Mingzhu; Yao, Wantong; Wang, Can; Li, Xin; Wang, Liying; Li, Zengxia; Shi, Yinghong; Qiu, Shuangjian; Fan, Jia; Zha, Xiliang

    2010-09-01

    DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

  13. Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

    PubMed

    Sekar, N; Veldhuis, J D

    2001-07-01

    -repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen

  14. Predicting the structures of complexes between phosphoinositide 3-kinase (PI3K) and romidepsin-related compounds for the drug design of PI3K/histone deacetylase dual inhibitors using computational docking and the ligand-based drug design approach.

    PubMed

    Oda, Akifumi; Saijo, Ken; Ishioka, Chikashi; Narita, Koichi; Katoh, Tadashi; Watanabe, Yurie; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2014-11-01

    Predictions of the three-dimensional (3D) structures of the complexes between phosphoinositide 3-kinase (PI3K) and two inhibitors were conducted using computational docking and the ligand-based drug design approach. The obtained structures were refined by structural optimizations and molecular dynamics (MD) simulations. The ligands were located deep inside the ligand binding pocket of the p110α subunit of PI3K, and the hydrogen bond formations and hydrophobic effects of the surrounding amino acids were predicted. Although rough structures were obtained for the PI3K-inhibitor complexes before the MD simulations, the refinement of the structures by these simulations clarified the hydrogen bonding patterns of the complexes.

  15. Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

    PubMed Central

    Kanda, Shigeru; Kanetake, Hiroshi; Miyata, Yasuyoshi

    2007-01-01

    Abstract The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells.LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis.Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation.Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells. PMID:17521372

  16. Auxin as an inducer of asymmetrical division generating the subsidiary cells in stomatal complexes of Zea mays.

    PubMed

    Livanos, Pantelis; Giannoutsou, Eleni; Apostolakos, Panagiotis; Galatis, Basil

    2015-01-01

    The data presented in this work revealed that in Zea mays the exogenously added auxins indole-3-acetic acid (IAA) and 1-napthaleneacetic acid (NAA), promoted the establishment of subsidiary cell mother cell (SMC) polarity and the subsequent subsidiary cell formation, while treatment with auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 1-napthoxyacetic acid (NOA) specifically blocked SMC polarization and asymmetrical division. Furthermore, in young guard cell mother cells (GMCs) the PIN1 auxin efflux carriers were mainly localized in the transverse GMC faces, while in the advanced GMCs they appeared both in the transverse and the lateral ones adjacent to SMCs. Considering that phosphatidyl-inositol-3-kinase (PI3K) is an active component of auxin signal transduction and that phospholipid signaling contributes in the establishment of polarity, treatments with the specific inhibitor of the PI3K LY294002 were carried out. The presence of LY294002 suppressed polarization of SMCs and prevented their asymmetrical division, whereas combined treatment with exogenously added NAA and LY294002 restricted the promotional auxin influence on subsidiary cell formation. These findings support the view that auxin is involved in Z. mays subsidiary cell formation, probably functioning as inducer of the asymmetrical SMC division. Collectively, the results obtained from treatments with auxin transport inhibitors and the appearance of PIN1 proteins in the lateral GMC faces indicate a local transfer of auxin from GMCs to SMCs. Moreover, auxin signal transduction seems to be mediated by the catalytic function of PI3K.

  17. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.

    PubMed Central

    Kotani, K; Yonezawa, K; Hara, K; Ueda, H; Kitamura, Y; Sakaue, H; Ando, A; Chavanieu, A; Calas, B; Grigorescu, F

    1994-01-01

    Insulin, IGF-1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced membrane ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, membrane ruffling. Images PMID:8194523

  18. TNF-α induces cytosolic phospholipase A2 expression via Jak2/PDGFR-dependent Elk-1/p300 activation in human lung epithelial cells.

    PubMed

    Yang, Chuen-Mao; Lee, I-Ta; Chi, Pei-Ling; Cheng, Shin-Ei; Hsiao, Li-Der; Hsu, Chih-Kai

    2014-03-15

    Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

  19. Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc

    NASA Technical Reports Server (NTRS)

    Chen, C.; Sytkowski, A. J.

    2001-01-01

    Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

  20. FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P(3) and phosphoinositide 3-kinase.

    PubMed

    Imeri, Faik; Blanchard, Olivier; Jenni, Aurelio; Schwalm, Stephanie; Wünsche, Christin; Zivkovic, Aleksandra; Stark, Holger; Pfeilschifter, Josef; Huwiler, Andrea

    2015-12-01

    Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.

  1. Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Fujibayashi, Tatsuya; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2015-10-01

    We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.

  2. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

    PubMed Central

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

  3. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

    PubMed

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

  4. The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

    PubMed

    Martin, Kayleigh J S; Muessel, Michelle J; Pullar, Christine E; Willars, Gary B; Wardlaw, Andrew J

    2015-01-01

    The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

  5. Vascular smooth muscle cell spreading onto fibrinogen is regulated by calpains and phospholipase C.

    PubMed

    Paulhe, F; Bogyo, A; Chap, H; Perret, B; Racaud-Sultan, C

    2001-11-09

    Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.

  6. Targeting phosphoinositide 3-kinase δ for allergic asthma.

    PubMed

    Rowan, Wendy C; Smith, Janet L; Affleck, Karen; Amour, Augustin

    2012-02-01

    Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3Kδ (phosphoinositide 3-kinase δ) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3Kδ inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3Kδ is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3Kδ should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3Kδ inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3Kδ inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma.

  7. Probing the PI3K/Akt/mTor pathway using 31P-NMR spectroscopy: routes to glycogen synthase kinase 3

    PubMed Central

    Phyu, Su M.; Tseng, Chih-Chung; Fleming, Ian N.; Smith, Tim A. D.

    2016-01-01

    Akt is an intracellular signalling pathway that serves as an essential link between cell surface receptors and cellular processes including proliferation, development and survival. The pathway has many downstream targets including glycogen synthase kinase3 which is a major regulatory kinase for cell cycle transit as well as controlling glycogen synthase activity. The Akt pathway is frequently up-regulated in cancer due to overexpression of receptors such as the epidermal growth factor receptor, or mutation of signalling pathway kinases resulting in inappropriate survival and proliferation. Consequently anticancer drugs have been developed that target this pathway. MDA-MB-468 breast and HCT8 colorectal cancer cells were treated with inhibitors including LY294002, MK2206, rapamycin, AZD8055 targeting key kinases in/associated with Akt pathway and the consistency of changes in 31P-NMR-detecatable metabolite content of tumour cells was examined. Treatment with the Akt inhibitor MK2206 reduced phosphocholine levels in MDA-MB-468 cells. Treatment with either the phosphoinositide-3-kinase inhibitor, LY294002 and pan-mTOR inhibitor, AZD8055 but not pan-Akt inhibitor MK2206 increased uridine-5′-diphosphate-hexose cell content which was suppressed by co-treatment with glycogen synthase kinase 3 inhibitor SB216763. This suggests that there is an Akt-independent link between phosphoinositol-3-kinase and glycogen synthase kinase3 and demonstrates the potential of 31P-NMR to probe intracellular signalling pathways. PMID:27811956

  8. Combination of PI3K/Akt/mTOR inhibitors and PDT in endothelial and tumor cells

    NASA Astrophysics Data System (ADS)

    Fateye, Babasola; Chen, Bin

    2011-02-01

    The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer. We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell line (PC3) and SV40-transformed mouse endothelial cell line (SVEC-40). Combination of PDT and BEZ 235 (BEZ), a pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of sub-lethal PDT in both cell lines. The combination of the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and LY rapidly after treatment. Compared to SVEC, PC3 cells are apoptosis-deficient and apoptosis was not significantly enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be enhanced by combination, depending on PI3K inhibitor and dose.

  9. Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway

    PubMed Central

    Lau, Yeh Siang; Ling, Wei Chih; Murugan, Dharmani; Kwan, Chiu Yin; Mustafa, Mohd Rais

    2015-01-01

    Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), NG-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NOx level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity. PMID:26133970

  10. HGF mediated upregulation of lipocalin 2 regulates MMP9 through nuclear factor-κB activation.

    PubMed

    Koh, Sung Ae; Lee, Kyung Hee

    2015-10-01

    Lipocalin 2 (LCN2) is a member of lipocalin family that binds and transports a small lipophilic ligand, sharing a highly conserved tertiary structure and can be found as a monomer, homodimer, heterodimer with matrix metalloproteinase 9 (MMP9). The high molecule LCN2/MMP9 complex was found in several cancer types. Yet, the mechanisms of regulation between LCN2 with MMP9 in tumorigenesis is unclear. The aims of the present study were to identify the function of LCN2 associated with MMP9 in gastric cancer growth and metastasis. First, we confirmed that the expression level of LCN2 and MMP9 was upregulated by hepatocyte growth factor (HGF). To identify the association pathway of HGF-induced LCN2, the cells were treated with PI3-kinase inhibitor (LY294002), or MEK inhibitor (PD098059), or p38 inhibitor (SB203580) and then analyzed using western blotting. The HGF-mediated LCN2 protein level was decreased with LY294002. Also, the HGF-mediated MMP9 was decreased with LY294002. The role for LCN2 with HGF mediated MMP9 was determined by knockdown of LCN2. LCN2-sh RNA cells showed a decreased level of HGF-mediated MMP9. The HGF-mediated LCN2 protein level was decreased with treatment of the NFκB inhibitor. We confirmed the role of HGF-mediated LCN2. HGF-mediated cell proliferation and in vitro invasion was decreased in LCN2 knockdown cell. In conclusion, the present study showed that LCN2 upregulated MMP9 through PI3K/AKT/NFκB pathway in gastric cancer. LCN2 has a role in cell proliferation and cell invasion in gastric cancer, which may be a possible target for developing gastric cancer therapy.

  11. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

    PubMed

    Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

    1995-10-24

    Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

  12. Class (I) Phosphoinositide 3-Kinases in the Tumor Microenvironment

    PubMed Central

    Gyori, David; Chessa, Tamara; Hawkins, Phillip T.; Stephens, Len R.

    2017-01-01

    Phosphoinositide 3-kinases (PI3Ks) are a diverse family of enzymes which regulate various critical biological processes, such as cell proliferation and survival. Class (I) PI3Ks (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) mediate the phosphorylation of the inositol ring at position D3 leading to the generation of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 can be dephosphorylated by several phosphatases, of which the best known is the 3-phosphatase PTEN (phosphatase and tensin homolog). The Class (I) PI3K pathway is frequently disrupted in human cancers where mutations are associated with increased PI3K-activity or loss of PTEN functionality within the tumor cells. However, the role of PI3Ks in the tumor stroma is less well understood. Recent evidence suggests that the white blood cell-selective PI3Kγ and PI3Kδ isoforms have an important role in regulating the immune-suppressive, tumor-associated myeloid cell and regulatory T cell subsets, respectively, and as a consequence are also critical for solid tumor growth. Moreover, PI3Kα is implicated in the direct regulation of tumor angiogenesis, and dysregulation of the PI3K pathway in stromal fibroblasts can also contribute to cancer progression. Therefore, pharmacological inhibition of the Class (I) PI3K family in the tumor microenvironment can be a highly attractive anti-cancer strategy and isoform-selective PI3K inhibitors may act as potent cancer immunotherapeutic and anti-angiogenic agents. PMID:28273837

  13. The citrus fruit flavonoid naringenin suppresses hepatic glucose production from Fao hepatoma cells.

    PubMed

    Purushotham, Aparna; Tian, Min; Belury, Martha A

    2009-02-01

    Hepatic gluconeogenesis is the major source of fasting hyperglycemia. Here, we investigated the role of the citrus fruit flavonoid naringenin, in the attenuation of hepatic glucose production from hepatoma (Fao) cells. We show that naringenin, but not its glucoside naringin, suppresses hepatic glucose production. Furthermore, unlike insulin-mediated suppression of hepatic glucose production, incubation of hepatocytes with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor Ly294002 had no effect on the ability of naringenin to suppress hepatic glucose production. Further, naringenin did not increase phosphorylation of Akt at Ser473 or, Thr308, indicating this down-stream target of PI3-kinase is also not a player in naringenin-mediated suppression of hepatic glucose production. Importantly, like the dimethylbiguanide, metformin, naringenin significantly decreased cellular ATP levels without increasing cell cytotoxicity. Together, these results suggest that the aglycone, naringenin, has a role in the attenuation of hyperglycemia and may exert this effect in a manner similar to the drug, metformin.

  14. The PI3K/Akt signaling pathway exerts effects on the implantation of mouse embryos by regulating the expression of RhoA

    PubMed Central

    LIU, LIYUAN; WANG, YINGXIONG; YU, QIUBO

    2014-01-01

    The aim of this study was to investigate whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway affects the implantation of mouse embryos by regulating the expression of RhoA. The expression of PI3K, Akt, phosphorylated (p-)Akt, phosphatase and tensin homolog (PTEN) and RhoA in the uterus of mice on day 5 of pregnancy (D5) and in pseudopregnant mice was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot analysis. A functional analysis of these genes was also performed by the intrauterine injection with the PI3K inhibitor, LY294002, on day 2 of pregnancy (D2). The expression levels of PI3K, p-Akt, RhoA at the implantation site were higher than those at the inter-implantation site in the endometrium; however, opposite effects were observed for PTEN expression. The expression levels of the above genes in the pseudopregnant group and in the group injected with the PI3K/Akt inhibitor, LY294002, were markedly lower than those in the pregnant group. Functional experiments revealed that the number of implantation sites had been significantly decreased (P<0.05) following the intrauterine injection of the PI3K inhibitor, LY294002, on day 2 of gestation compared with the contralateral injection of phosphate-buffered saline (PBS). These results suggest that the PI3K/Akt signaling pathway affects embryo implantation by regulating the expression of RhoA. PMID:24638941

  15. The PI3K signaling-mediated nitric oxide contributes to cardiovascular effects of angiotensin-(1-7) in the nucleus tractus solitarii of rats.

    PubMed

    Wu, Zhao-Tang; Ren, Chang-Zhen; Yang, Ya-Hong; Zhang, Ru-Wen; Sun, Jia-Cen; Wang, Yang-Kai; Su, Ding-Feng; Wang, Wei-Zhong

    2016-01-30

    Angiotensin-1-7 [Ang-(1-7)], acting via the Mas receptor in the central nervous system, is involved in the regulation of cardiovascular activity. Nitric oxide (NO) is implicated as an important modulator in the nucleus tractus solitarii (NTS), a key region involved in control of cardiovascular activity. The aim of the present study was to determine the role of phosphatidylinositol 3-kinase (PI3K) signaling in mediating the effect of Ang-(1-7) on NO generation in the NTS. In Sprague-Dawley rats, acute injection of Ang-(1-7) into the NTS significantly increased NO generation and neuronal/endothelial NO synthase (n/eNOS) activity, which were abolished by the selective Mas receptor antagonist d-Alanine-[Ang-(1-7)] (A-779), the PI3K inhibitor LY294002, or the Akt inhibitor triciribine (TCN). Western blotting analysis further demonstrated that Ang-(1-7) significantly increased levels of Akt/NOS phosphorylation in the NTS, and Ang-(1-7)-induced e/nNOS phosphorylation was antagonized by LY294002 or TCN. Furthermore, gene knockdown of PI3K by lentivirus containing small hairpin RNA in the NTS prevented the Ang-(1-7)-induced increases in NOS/Akt phosphorylation and NO production. The physiological (in vivo) experiments showed that pretreatment with the NOS inhibitor l-NAME, LY294002, or TCN abolished the decreases in blood pressure, heart rate, and renal sympathetic nerve activity induced by Ang-(1-7) injected into the NTS. Our findings suggest that nitric oxide release meditated by the Mas-PI3K-NOS signaling pathway is involved in the cardiovascular effects of Ang-(1-7) in the NTS.

  16. Dasatinib synergizes with both cytotoxic and signal transduction inhibitors in heterogeneous breast cancer cell lines--lessons for design of combination targeted therapy.

    PubMed

    Park, Brian J; Whichard, Zakary L; Corey, Seth J

    2012-07-01

    Molecularly targeted therapies have emerged as the leading theme in cancer therapeutics. Multi-cytotoxic drug regimens have been highly successful, yet many studies in targeted therapeutics have centered on a single agent. We investigated whether the Src/Abl kinase inhibitor dasatinib displays synergy with other agents in molecularly heterogeneous breast cancer cell lines. MCF-7, SKBR-3, and MDA-MB-231 display different signaling and gene signatures profiles due to expression of the estrogen receptor, ErbB2, or neither. Cell proliferation was measured following treatment with dasatinib±cytotoxic (paclitaxel, ixabepilone) or molecularly targeted agents (tamoxifen, rapamycin, sorafenib, pan PI3K inhibitor LY294002, and MEK/ERK inhibitor U0126). Dose-responses for single or combination drugs were calculated and analyzed by the Chou-Talalay method. The drugs with the greatest level of synergy with dasatinib were rapamycin, ixabepilone, and sorafenib, for the MDA-MB-231, MCF-7, and SK-BR-3 cell lines respectively. However, dasatinib synergized with both cytotoxic and molecularly targeted agents in all three molecularly heterogeneous breast cancer cell lines. These results suggest that effectiveness of rationally designed therapies may not entirely rest on precise identification of gene signatures or molecular profiling. Since a systems analysis that reveals emergent properties cannot be easily performed for each cancer case, multi-drug regimens in the near future will still involve empirical design.

  17. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.

    PubMed

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

    2003-07-01

    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  18. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

    PubMed Central

    Shepherd, P R; Withers, D J; Siddle, K

    1998-01-01

    Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

  19. LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

    SciTech Connect

    Tong, W.-G.; Ding, X.-Z.; Talamonti, Mark S.; Bell, Richard H.; Adrian, Thomas E. . E-mail: tadrian@northwestern.edu

    2005-09-30

    We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.

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

    PubMed Central

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

    2014-01-01

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

  1. Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg.

    PubMed

    Murakami, Shigefumi; Sakurai-Yageta, Mika; Maruyama, Tomoko; Murakami, Yoshinori

    2014-01-01

    CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently downregulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

  2. Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg.

    PubMed

    Murakami, Shigefumi; Sakurai-Yageta, Mika; Maruyama, Tomoko; Murakami, Yoshinori

    2014-01-01

    CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently down-regulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

  3. Evaluation of Potential Ionizing Irradiation Protectors and Mitigators Using Clonogenic Survival of Human Umbilical Cord Blood Hematopoietic Progenitor Cells

    PubMed Central

    Goff, Julie P.; Shields, Donna S.; Wang, Hong; Skoda, Erin M.; Sprachman, Melissa M.; Wipf, Peter; Garapati, Venkata Krishna; Atkinson, Jeffrey; London, Barry; Lazo, John S.; Kagan, Valerian; Epperly, Michael W.; Greenberger, Joel S.

    2013-01-01

    We evaluated the use of colony formation (CFU-GM, BFU-E, and CFU-GEMM) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. Each of 11 compounds was added before (protection) or after (mitigation) ionizing irradiation including: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor (LY294002), TPP-imidazole fatty acid, (TPP-IOA), the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propanolol, and the ATP sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs, XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs that were effective in murine assays: TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide showed no significant protection or mitigation in human CB assays. These data support testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, reducing the need for animal experiments. PMID:23933481

  4. PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Dasgupta, Arindam; Jung, Kyung-Jin; Um, Jee-Hyun; Burke, Aileen; Park, Hyeon Ung; Brady, John N

    2008-01-20

    The phosphatidylinositol-3-kinase (PI3K) and AKT (protein kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. We demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase-9 is activated. Pretreatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-kappaB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy.

  5. Suppression of Mcl-1 induces apoptosis in mouse peritoneal macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Wang, Fei-Yu; Wang, Xin-Min; Wang, Chan; Wang, Xiao-Fang; Zhang, Yu-Qing; Wu, Jiang-Dong; Wu, Fang; Zhang, Wan-Jiang; Zhang, Le

    2016-04-01

    The effect of myeloid cell leukemia-1 (Mcl-1) inhibition on apoptosis of peritoneal macrophages in mice infected with Mycobacterium tuberculosis was investigated and the primary signaling pathway associated with the transcriptional regulation of Mcl-1 was identified. Real-time PCR and western blotting indicated that Mcl-1 transcript and protein expression are upregulated during infection with virulent M. tuberculosis H37Rv and Xinjiang strains but not with attenuated M. tuberculosis strain H37Ra or Bacillus Calmette-Guérin. Mcl-1 transcript and protein expression were downregulated by specific inhibitors of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways (AG490, PD98059 and LY294002, respectively). The strongest inhibitor of Mcl-1 expression was PD98059, the MAPK inhibitor. Flow cytometry demonstrated that the rate of apoptosis in peritoneal macrophages is significantly higher in mice infected with M. tuberculosis and the rate of apoptosis is correlated with the virulence of the strain of M. tuberculosis. Apoptosis was found to be upregulated by AG490, PD98059 and LY294002, whereas inhibition of the MAPK pathway sensitized the infected macrophages to apoptosis. Taken together, these results suggest that specific downregulation of Mcl-1 significantly increases apoptosis of peritoneal macrophages and that the MAPK signaling pathway is the primary mediator of Mcl-1 expression.

  6. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    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 (2.5-20 μM) 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.

  7. Acquired resistance to oestrogen deprivation: role for growth factor signalling kinases/oestrogen receptor cross-talk revealed in new MCF-7X model.

    PubMed

    Staka, Cindy M; Nicholson, Robert I; Gee, Julia M W

    2005-07-01

    In vitro models of long-term oestrogen deprivation utilise increased oestrogen receptor (ER) and are oestrogen hypersensitive, with emerging evidence that growth factor signalling contributes and interacts with ER. However, such models are commonly derived in the presence of serum growth factors that may force the resistance mechanism. Our new in vitro model, MCF-7X, has thus been developed under conditions of both oestrogen and growth factor depletion. ER expression, serine 118 phosphorylation on this receptor and its transcriptional activity were modestly increased compared to the parental MCF-7 cells, although MCF-7X cells were not oestrogen hypersensitive. Faslodex (0.1 microM) partially decreased ER and its transcriptional activity, with associated decreases in serine 118 phosphorylation. Faslodex inhibited MCF-7X growth by 50% for 10 weeks. Classical growth factor receptors did not impact on MCF-7X growth and only a modest contribution for MAP kinase was revealed using PD98059 (25 microM; 35% inhibition for 3 weeks). However, the phosphatidylinositol-3-OH (PI3)-kinase inhibitor LY294002 (5 microM) inhibited MCF-7X growth by 65% for 10 weeks. In contrast to PD98059, LY294002 also partially-inhibited ER transcriptional activity and decreased serine 167 ER phosphorylation. Co-treatment with faslodex plus LY294002 to decrease activity of both serine 118 and 167 proved superior vs the single agents in decreasing ER transcriptional activity and MCF-7X growth (90% inhibition for 25 weeks). However, triple treatment including PD98059 was required to prevent resistance in MCF-7X, an event dependent on maximal depletion of serine 118 phosphorylation and ER transcriptional activity. Kinases clearly contribute in resistance to oestrogen deprivation, cross-talking with ER signalling via AF-1 phosphorylation. While inhibiting each pathway has potential to treat this state, combined therapy targeting all regulators of ER phosphorylation may be required to block subsequent

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

    SciTech Connect

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

    2014-07-18

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

  9. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

    PubMed

    Park, Dae-Won; Kim, Jae-Ryong; Kim, Seong-Yong; Sonn, Jong-Kyung; Bang, Ok-Sun; Kang, Shin-Sung; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-02-15

    The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

  10. Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy.

    PubMed

    Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

    2015-01-01

    Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases.

  11. Two distinct functions for PI3-kinases in macropinocytosis

    PubMed Central

    Hoeller, Oliver; Bolourani, Parvin; Clark, Jonathan; Stephens, Len R.; Hawkins, Phillip T.; Weiner, Orion D.; Weeks, Gerald; Kay, Robert R.

    2013-01-01

    Summary Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins. PMID:23843627

  12. Inhibition of PI-3 kinase for treating respiratory disease: good idea or bad idea?

    PubMed

    Thomas, Matt; Owen, Charles

    2008-06-01

    Inhibition of one or more members of the phosphoinositide 3-kinase (PI3K) family for the treatment of respiratory diseases remains the goal of many pharmaceutical companies over the past 20 years. Here we briefly review the PI3K family, then focus on the assessment of each isoform as a drug discovery target. The rationale for PI3Kalpha inhibition in the treatment of lung cancer, and PI3Kbeta inhibitors in pulmonary thrombotic processes, are balanced with a potential side effect profile affecting metabolism and/or foetal development. Roles for PI3Kdelta in inflammatory lung diseases and PI3Kgamma in asthma are weighed against the consequences of manipulating key immune cell populations. We also discuss the current status and future potential of PI3K inhibitors in respiratory disease.

  13. Systemic application of 3-methyladenine markedly inhibited atherosclerotic lesion in ApoE−/− mice by modulating autophagy, foam cell formation and immune-negative molecules

    PubMed Central

    Dai, Shen; Wang, Bo; Li, Wen; Wang, Liyang; Song, Xingguo; Guo, Chun; Li, Yulan; Liu, Fengming; Zhu, Faliang; Wang, Qun; Wang, Xiaoyan; Shi, Yongyu; Wang, Jianing; Zhao, Wei; Zhang, Lining

    2016-01-01

    A growing body of evidence demonstrates that autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of atherosclerosis and has become a potential therapeutic target. Here we tested the effect of two inhibitors of phosphatidylinositol 3-kinase, 3-methyladenine (3-MA) and 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), commonly used as inhibitors of autophagy, in atherosclerosis in apolipoprotein E−/− mice. Systemic application of 3-MA but not LY294002 markedly reduced the size of atherosclerotic plaque and increased the stability of lesions in high-fat diet-fed mice as compared with controls. Furthermore, 3-MA had multiple atheroprotective effects, including modulating macrophage autophagy and foam cell formation and altering the immune microenvironment. Long-term treatment with 3-MA promoted oxidized low-density lipoprotein (oxLDL)-induced macrophage autophagy and suppressed foam cell formation and cell viability in vitro. Furthermore, systemic application of 3-MA promoted lipid droplet breakdown and decreased apoptosis, most likely associated with autophagy. 3-MA treatment strikingly enhanced the expression of immune-negative molecules such as interleukin 10 (IL-10), transforming growth factor β and IL-35, as well as forkhead box P3 (Foxp3), the specific transcriptional factor for regulatory T cells, but did not affect the level of proinflammatory cytokines in the arterial wall. We provide strong evidence for the potential therapeutic benefit of 3-MA in inhibiting atherosclerosis development and improving plaque stability. PMID:27906187

  14. G-protein-coupled receptor 30-mediated antiapoptotic effect of estrogen on spinal motor neurons following injury and its underlying mechanisms.

    PubMed

    Chen, Jingyu; Hu, Rong; Ge, Hongfei; Duanmu, Wangsheng; Li, Yuhong; Xue, Xingseng; Hu, Shengli; Feng, Hua

    2015-08-01

    Spinal cord injury (SCI) may result in severe dysfunction of motor neurons. G-protein-coupled receptor 30 (GPR30) expression in the motor neurons of the ventral horn of the spinal cord mediates neuroprotection through estrogen signaling. The present study explored the antiapoptotic effect of estrogen, mediated by GPR30 following SCI, and the mechanisms underlying this effect. Spinal motor neurons from rats were cultured in vitro in order to establish cell models of oxygen and glucose deprivation (OGD). The effects of estrogen, the estrogen agonist, G1, and the estrogen inhibitor, G15, on motor neurons were observed using MTT assays. The effects of E2, G1 and G15 on spinal motor neuron apoptosis following OGD, were detected using flow cytometry. The role of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) inhibitor, LY294002, was also determined using flow cytometry. Rat SCI models were established. E2, G1 and E2+LY294002 were administered in vivo. Motor function was scored at 3, 7, 14, 21 and 28 d following injury, using Basso-Beattie-Bresnahan (BBB) standards. Cell activity in the estrogen and G1 groups was higher than that in the solvent group, whereas cell activity in the E2+G15 group was lower than that in the E2 group (P<0.05). Following OGD, the proportion of apoptotic cells significantly increased (P<0.05). The proportion in the estrogen group was significantly lower than that in the solvent group, whereas the proportion of apoptotic cells in the E2+G15 and E2+LY294002 groups was higher than that in the E2 group (P<0.05). Treatment with E2 and G1 led to upregulation of P-Akt expression in normal cells and post-OGD cells. The BBB scores of rats in the E2 and G1 groups were higher than those in the placebo group (P<0.05). The BBB scores of the E2+LY294002 group were lower than those of the E2 group (P<0.05). Estrogen thus appears to exert a protective effect on spinal motor neurons following OGD, via GPR30. The PI3K/Akt pathway may be one of those

  15. Idelalisib: Targeting the PI3 Kinase Pathway in Non-Hodgkin Lymphoma.

    PubMed

    Sujobert, Pierre; Rioufol, Catherine; Salles, Gilles A

    2016-01-01

    Based on substantial preclinical rationale, the restricted hematopoietic expression of the δ isoform of the phosphatidylinositol 3-kinase represents an attractive therapeutic target in B-cell malignancies. Its inhibition results in a direct antiproliferative effect on tumor cells as well as several modifications of their cellular microenvironment, all accounting for the potential therapeutic interest. Idelalisib, the first-in-class phosphatidylinositol 3-kinase δ-specific inhibitor, was developed in patients with B-cell lymphomas and chronic lymphocytic leukemia. Early clinical results demonstrated a potent antitumor effect across different subtypes of indolent and mantle cell lymphomas (where response duration was short). Adverse events, including transaminitis, neutropenia, pneumonitis, and diarrhea, were observed. A pivotal phase II study in patients with double refractory disease showed a 57% response rate, with response lasting for about 1 year, leading to market approval of the drug in the United States and Europe. Further developments of idelalisib combinations will contribute to delineate the position of this drug in the therapeutic strategy of indolent lymphomas.

  16. Pathophysiological roles of Pim-3 kinase in pancreatic cancer development and progression.

    PubMed

    Li, Ying-Yi; Mukaida, Naofumi

    2014-07-28

    Pim-3 is a member of the provirus integration site for Moloney murine leukemia virus (Pim) family proteins that exhibit serine/threonine kinase activity. Similar to the other Pim kinases (Pim-1 and Pim-2), Pim-3 is involved in many cellular processes, including cell proliferation, survival, and protein synthesis. Although Pim-3 is expressed in normal vital organs, it is overexpressed particularly in tumor tissues of endoderm-derived organs, including the liver, pancreas, and colon. Silencing of Pim-3 expression can retard in vitro cell proliferation of hepatocellular, pancreatic, and colon carcinoma cell lines by promoting cell apoptosis. Pim-3 lacks the regulatory domains similarly as Pim-1 and Pim-2 lack, and therefore, Pim-3 can exhibit its kinase activity once it is expressed. Pim-3 expression is regulated at transcriptional and post-transcriptional levels by transcription factors (e.g., Ets-1) and post-translational modifiers (e.g., translationally-controlled tumor protein), respectively. Pim-3 could promote growth and angiogenesis of human pancreatic cancer cells in vivo in an orthotopic nude mouse model. Furthermore, a Pim-3 kinase inhibitor inhibited cell proliferation when human pancreatic cancer cells were injected into nude mice, without inducing any major adverse effects. Thus, Pim-3 kinase may serve as a novel molecular target for developing targeting drugs against pancreatic and other types of cancer.

  17. Fibroblast growth factor 4-induced migration of porcine trophectoderm cells is mediated via the AKT cell signaling pathway.

    PubMed

    Jeong, Wooyoung; Lee, Jieun; Bazer, Fuller W; Song, Gwonhwa; Kim, Jinyoung

    2016-01-05

    During early pregnancy, a well-coordinated communication network between the conceptus and maternal uterus is especially crucial in pigs in which there is a protracted pre-attachment phase prior to implantation. This network is regulated by an astonishing number of molecules such as growth factors. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor that elicits diverse biological actions on various types of cells and tissues. In pigs, FGF4 and its receptors are expressed in the uterine endometrium and conceptus during early pregnancy, but less is known about the FGF4-mediated regulation of conceptus growth during peri-implantation period of pregnancy. Therefore, the aims of the present study were to investigate: 1) expression of endometrial FGF4 mRNA during early pregnancy; 2) up-regulation of FGF receptor expression in porcine trophectoderm (pTr) cells in response to FGF4; and 3) FGF-induced intracellular signaling and cellular activities in pTr cells. In vitro cultured pTr cells incubated with different concentrations of recombinant FGF4 (0-50 ng/ml) responded with a dose-dependent increase in AKT phosphorylation of 2.9-fold at 20 ng/ml FGF4. Within 30 min after treatment with 20 ng/ml FGF4, the abundances of p-AKT, p-P90RSK and p-RPS6 proteins increased 2.1-, 5.2- and 3.2-fold, respectively, and then returned to basal levels by 120 min. To ensure that the stimulatory effect of FGF4 on AKT signaling was p-AKT-dependent, pTr cells were pre-incubated with an AKT inhibitor (LY294002) for 1 h prior to FGF4 treatment. 20 μM of LY294002 decreased FGF4-induced p-AKT, p-P90RSK and p-RPS6 proteins. Immunofluorescence analyses revealed that p-RPS6 proteins were abundant within the cytoplasm of FGF4-treated cells, but present at basal levels in the presence of LY294002. Furthermore, FGF4 increased migration of pTr cells and LY294002 significantly reduced this effect. Results of the present study suggest that activation of the FGF receptor(s) on trophectoderm

  18. A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells

    SciTech Connect

    Iqbal, Mohd S.; Tsuyama, Naohiro; Obata, Masanori; Ishikawa, Hideaki

    2010-02-12

    Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21{sup Cip1} proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

  19. Modulation of the cellular content of metabolites in adipocytes by insulin.

    PubMed

    Qiao, Yuhang; Tomonaga, Shozo; Matsui, Tohru; Funaba, Masayuki

    2016-03-15

    Although the insulin-mediated cell signaling pathway has been extensively examined, changes in the cellular content of metabolites currently remain unclear. We herein examined metabolite contents in 3T3-L1 adipocytes treated with insulin using a metabolomic analysis. Fifty-four compounds were detected, and the contents of metabolites from the citric acid cycle increased in response to the insulin treatment for 4 h, which was sensitive to U0126 and LY294002, inhibitors for mitogen-activated protein kinase kinase-1 and phosphoinositide 3-kinase, respectively. The cellular contents of fumaric acid and malic acid were increased more by insulin than those of citric acid and succinic acid. Time-course changes in metabolites from the citric acid cycle exhibited oscillations with a 2-h cycle. A metabolic pathway analysis also indicated that insulin affected the metabolism of alanine, aspartate and glutamate, as well as that of arginine and proline. The contents of free amino acids were slightly decreased by the insulin treatment, while the co-treatment with U0126 and LY294002 abrogated these insulin-mediated decreases. The present study revealed the unexpected accumulation of citric acid cycle metabolites in adipocytes by insulin. Our results indicate the usefulness of metabolomic analyses for obtaining a more comprehensive understanding of the regulation of metabolic pathways in cell-culture systems.

  20. Role of IRS-4 in PI3-K activation by insulin in HepG2 cells, modulation by Angiotensin II.

    PubMed

    Villarreal, Rodrigo Sebastián; Forneris, Myriam Liliana; Uranga, Romina María; Salvador, Gabriela Alejandra; Ciuffo, Gladys María

    2010-04-09

    Insulin receptor substrate-4 (IRS-4) has a limited tissue expression and its modulation by tyr-phosphorylation is still controversial. We evaluated the participation of IRS-4 in the cross-talk between Angiotensin II (Ang II) and Insulin (Ins) receptors in HepG2 cells. Ins (10(-7)M) induced tyr-phosphorylation of IRS-4 (maximal at 5 min), an effect potentiated by Ang II AT(1) receptors. Phosphatydilinositol-3 kinase (PI3-K) inhibitors Wortmanin or LY294002 reduced Ang II effect on tyr-phosphorylation of IRS-4 to a level comparable to that of Ins alone. Physical association between IRS-4 substrate and PI3-K was demonstrated by co-immunoprecipitation. Recruitment of PI3-K by IRS-4 was induced by Ins (10(-7)M, 5 min) not by Ang II (10(-7)M) and this was inhibited by Wortmanin and LY294002. Ang II did not modify either the association or activation of PI3-K in immunocomplexes. The present data provide novel evidence of IRS-4 phosphorylation mediated by Ins, an effect modulated by Ang II. We report also Ins-induced PI3-K activation mediated by IRS-4. Our findings suggest a role for IRS-4 as a docking protein in the Ins signaling pathway that involves PI3-K association and activation. The present data suggest a possible participation of IRS-4 in cell proliferation Ins-induced.

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

    PubMed

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

    2016-01-01

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

  2. Increased basal level of Akt-dependent insulin signaling may be responsible for the development of insulin resistance

    PubMed Central

    Liu, Hui-Yu; Hong, Tao; Wen, Ge-Bo; Han, Jianmin; Zuo, Degen; Liu, Zhenqi

    2009-01-01

    A majority of subjects with insulin resistance and hyperinsulinemia can maintain their blood glucose levels normal for the whole life presumably through protein kinase B (Akt)-dependent insulin signaling. In this study, we found that the basal Akt phosphorylation level was increased in liver and gastrocnemius of mice under the high-fat diet (HFD). Levels of mitochondrial DNA and expression of some mitochondrion-associated genes were decreased by the HFD primarily in liver. Triglyceride content was increased in both liver and gastrocnemius by the HFD. Oxidative stress was induced by the HFD in both liver and gastrocnemius. Insulin sensitivity was decreased by the HFD. All of these changes were largely or completely reversed by treatment of animals with the phosphatidylinositol 3-kinase inhibitor LY-294002 during the time when animals usually do not eat. Consequently, the overall insulin sensitivity was increased by treatment with LY-294002. Together, our results indicate that increased basal Akt-dependent insulin signaling suppresses mitochondrial production, increases ectopic fat accumulation, induces oxidative stress, and desensitizes insulin signaling in subjects with insulin resistance and hyperinsulinemia. PMID:19638508

  3. Impact of E6-associated protein on the proliferation and invasion of prostate cancer cells in bone metastasis

    PubMed Central

    Zhang, Liyan; Hu, Xiaoguang; Chen, Jiying; Fu, Guilian

    2015-01-01

    Purpose: To understand E6 associated protein (E6-AP)’s influence on prostate cancer cell proliferation and infiltration, thus providing the theoretical basis for developing therapeutic drugs for prostate cancer metastasis to the bone. Methods: Electroporation was performed to introduce linear regulatory plasmid PrevTet-off-in and conjugative plasmid PrevTRE2-flag-E6AP into prostate cancer cell line to establish wild-type E6-AP over-expressing transgenic LNCaP cell line; Western blot assay was adopted to examine expression levels of E6-AP, mammalian target of rapamycin (mTOR), protein kinase B (Akt), and phosphoinositide 3-kinase (PI3K); PI3K inhibitor LY294002 was applied to all the cells and MTT assay was used to measure cell proliferation; Matrigel invasion chamber assay was adopted to detect cancer cell migration and invasion. Results: Stably transfected LNCaP cells that over expressed E6-AP had higher expression levels of PI3K, Akt, and mTOR than control LNCaP cells; MTT assay showed that E6-AP-LNCaP cells were more responsive to the inhibitory effect of LY294002; Matrigel invasion chamber assay revealed increased cell crawling and adhesiveness of E6-AP-LNCaP cells. Conclusion: Stable over-expression of E6-AP increases the proliferation and invasion of LNCaP cells. PMID:26261538

  4. Differential roles of MAPK-Erk1/2 and MAPK-p38 in insulin or insulin-like growth factor-I (IGF-I) signaling pathways for progesterone production in human ovarian cells.

    PubMed

    Seto-Young, D; Avtanski, D; Varadinova, M; Park, A; Suwandhi, P; Leiser, A; Parikh, G; Poretsky, L

    2011-06-01

    Insulin and insulin like-growth factor-I (IGF-I) participate in the regulation of ovarian steroidogenesis. In insulin resistant states ovaries remain sensitive to insulin because insulin can activate alternative signaling pathways, such as phosphatidylinositol-3-kinase (PI-3 kinase) and mitogen-activated protein-kinase (MAPK) pathways, as well as insulin receptors and type 1 IGF receptors. We investigated the roles of MAPK-Erk1/2 and MAPK-p38 in insulin and IGF-I signaling pathways for progesterone production in human ovarian cells. Human ovarian cells were cultured in tissue culture medium in the presence of varying concentrations of insulin or IGF-I, with or without PD98059, a specific MAPK-Erk1/2 inhibitor, with or without SB203580, a specific MAPK-p38 inhibitor or with or without a specific PI-3-kinase inhibitor LY294002. Progesterone concentrations were measured using radioimmunoassay. PD98059 alone stimulated progesterone production in a dose-dependent manner by up to 65% (p<0.001). Similarly, LY294002 alone stimulated progesterone production by 13-18% (p<0.005). However, when used together, PD98059 and LY294002 inhibited progesterone production by 17-20% (p<0.001). SB203580 alone inhibited progesterone production by 20-30% (p<0.001). Insulin or IGF-I alone stimulated progesterone production by 40-60% (p<0.001). In insulin studies, PD98059 had no significant effect on progesterone synthesis while SB203580 abolished insulin-induced progesterone production. Either PD98059 or SB203580 abolished IGF-I-induced progesterone production. Both MAPK-Erk1/2 and MAPK-p38 participate in IGF-I-induced signaling pathways for progesterone production, while insulin-induced progesterone production requires MAPK-p38, but not MAPK-Erk1/2. These studies provide further evidence for divergence of insulin and IGF-I signaling pathways for human ovarian cell steroidogenesis.

  5. Selective Inhibition of Phosphoinositide 3-Kinase p110α Preserves Lymphocyte Function*

    PubMed Central

    So, Lomon; Yea, Sung Su; Oak, Jean S.; Lu, Mengrou; Manmadhan, Arun; Ke, Qiao Han; Janes, Matthew R.; Kessler, Linda V.; Kucharski, Jeff M.; Li, Lian-Sheng; Martin, Michael B.; Ren, Pingda; Jessen, Katti A.; Liu, Yi; Rommel, Christian; Fruman, David A.

    2013-01-01

    Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors. PMID:23275335

  6. Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

    PubMed

    So, Lomon; Yea, Sung Su; Oak, Jean S; Lu, Mengrou; Manmadhan, Arun; Ke, Qiao Han; Janes, Matthew R; Kessler, Linda V; Kucharski, Jeff M; Li, Lian-Sheng; Martin, Michael B; Ren, Pingda; Jessen, Katti A; Liu, Yi; Rommel, Christian; Fruman, David A

    2013-02-22

    Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

  7. PIK3CA is implicated as an oncogene in ovarian cancer

    SciTech Connect

    Shayesteh, Laleh; Lu, Yiling; Kuo, Wen-Lin; Baldocchi, Russell; Godfrey, Tony; Collins, Colin; Pinkel, Daniel; Powell, Bethan; Mills,Gordon B.; Gray, Joe W.

    1998-03-25

    Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about its molecular aetiology. Studies using comparative genomic hybridization (CGH) have revealed several regions of recurrent, abnormal, DNA sequence copy number that may encode genes involved in the genesis or progression of the disease. One region at 3q26 found to be increased in copy number in approximately 40 percent of ovarian and other cancers contains PIK3CA, which encodes the p110 a catalytic subunit of phosphatidylinositol 3-kinase(PI3-kinase). The association between PIK3CA copy number and PI3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI3-kinase mediated signaling. These include proliferation, glucose transport and catabolism, cell adhesion, apoptosis, RAS signaling and oncogenic transformation. In addition, downstream effectors of PI3-kinase,AKT1 and AKT2, have been found to be amplified or activated in human tumors, including ovarian cancer. We show here that PIK3CA is frequently increased in copy number in ovarian cancers, that the increased copy number is associated with increased PIK3CA transcription, p110 a protein expression and PI3-kinase activity and that treatment with the PI3-kinase inhibitor LY294002 decreases proliferation and increases apoptosis. Our observations suggest PIK3CA is an oncogene that has an important role in ovarian cancer.

  8. Phosphoinositide 3-kinase signaling in the vertebrate retina

    PubMed Central

    Rajala, Raju V. S.

    2010-01-01

    The phosphoinositide (PI) cycle, discovered over 50 years ago by Mabel and Lowell Hokin, describes a series of biochemical reactions that occur on the inner leaflet of the plasma membrane of cells in response to receptor activation by extracellular stimuli. Studies from our laboratory have shown that the retina and rod outer segments (ROSs) have active PI metabolism. Biochemical studies revealed that the ROSs contain the enzymes necessary for phosphorylation of phosphoinositides. We showed that light stimulates various components of the PI cycle in the vertebrate ROS, including diacylglycerol kinase, PI synthetase, phosphatidylinositol phosphate kinase, phospholipase C, and phosphoinositide 3-kinase (PI3K). This article describes recent studies on the PI3K-generated PI lipid second messengers in the control and regulation of PI-binding proteins in the vertebrate retina. PMID:19638643

  9. Phosphoinositide 3-kinase signaling is critical for ErbB3-driven breast cancer cell motility and metastasis

    PubMed Central

    Smirnova, Tatiana; Zhou, Zhen Ni; Flinn, Rory J.; Wyckoff, Jeffrey; Boimel, Pamela J.; Pozzuto, Maria; Coniglio, Salvatore J.; Backer, Jonathan M.; Bresnick, Anne R.; Condeelis, John S.; Hynes, Nancy E.; Segall, Jeffrey E.

    2011-01-01

    Many malignancies show increased expression of the EGF receptor family member ErbB3 (HER3). ErbB3 binds beta-1 (HRGβ1), and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGFR (HER1), enhancing phosphorylation of specific C terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of PI3-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGβ1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGβ1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, while mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor. PMID:21725367

  10. Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma

    PubMed Central

    Singh, Alok R.; Joshi, Shweta; Burgoyne, Adam M.; Sicklick, Jason K.; Ikeda, Sadakatsu; Kono, Yuko; Garlich, Joseph R.; Morales, Guillermo A.; Durden, Donald L.

    2017-01-01

    Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo. Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual "first-in-class" PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. PMID:27496136

  11. Supramolecular nanoparticles that target phosphatidylinositol-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy

    PubMed Central

    Kulkarni, Ashish A.; Roy, Bhaskar; Rao, Poornima S.; Wyant, Gregory A.; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M.; Sengupta, Shiladitya

    2013-01-01

    The centrality of phosphatidylinositol-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intra-tumoral concentration and an insulin resistance ‘class effect’. The current study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG. The supramolecular nanoparticles that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-RasLSL/+/Ptenfl/fl ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the supramolecular nanoparticles highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the supramolecular nanoparticles exerted a temporally-sustained inhibition of phosphorylation of Akt, mTOR, S6K and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of supramolecular nanoparticles abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer treatment

  12. Supramolecular nanoparticles that target phosphoinositide-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy.

    PubMed

    Kulkarni, Ashish A; Roy, Bhaskar; Rao, Poornima S; Wyant, Gregory A; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M; Sengupta, Shiladitya

    2013-12-01

    The centrality of phosphoinositide-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intratumoral concentration, and an insulin resistance "class effect." This study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polythylene glycol)]. The supramolecular nanoparticles (SNP) that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-Ras(LSL/+)/Pten(fl/fl) ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the SNPs highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the SNPs exerted a temporally sustained inhibition of phosphorylation of Akt, mTOR, S6K, and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of SNPs abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer

  13. Diosgenin inhibits melanogenesis through the activation of phosphatidylinositol-3-kinase pathway (PI3K) signaling.

    PubMed

    Lee, Jongsung; Jung, Kwangseon; Kim, Yeong Shik; Park, Deokhoon

    2007-06-27

    An increased level of melanin is characteristic of a large number of skin diseases, including acquired hyperpigmentation conditions such as melasma, post inflammatory melanoderma, and solar lentigo. Thus, there is an increasing need for the development of depigmenting agents. In order to evaluate the depigmenting capacity of diosgenin and elucidate its mechanism of action, several experiments were performed in B16 melanoma cells. Melanin content and Western blots for proteins that are involved in melanogenesis were assessed in this study. The melanin content was significantly inhibited by diosgenin. To clarify the mechanism of the depigmenting property of diosgenin, we examined the involvement of diosgenin in the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, diosgenin inhibited the reduction of Akt and GSK 3beta phosphorylation induced by LY294,002, a PI3K inhibitor. In accordance with this result, production levels of MITF (microphthalmia-associated transcription factor) and tyrosinase were increased by diosgenin. These data suggest that diosgenin inhibits melanogenesis through the activation of the PI3K pathway. This suggestion was further confirmed by the fact that the increased production level of melanin by LY294,002 was reduced by diosgenin in B16 melanoma cells. Our study shows that diosgenin inhibits melanogenesis by activating the PI3K pathway, and also suggests that diosgenin may be an effective inhibitor of hyperpigmentation.

  14. Phosphatidylinositol 3-Kinase γ is required for the development of experimental cerebral malaria.

    PubMed

    Lacerda-Queiroz, Norinne; Brant, Fatima; Rodrigues, David Henrique; Vago, Juliana Priscila; Rachid, Milene Alvarenga; Sousa, Lirlândia Pires; Teixeira, Mauro Martins; Teixeira, Antonio Lucio

    2015-01-01

    Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.

  15. Polycystin-1 Induces Resistance to Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Boca, Manila; Distefano, Gianfranco; Boletta, Alessandra; Qian, Feng; Bhunia, Anil K.; Germino, Gregory G.

    2006-01-01

    Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K β activity in PC-1- expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease. PMID:16452497

  16. GM3 suppresses anchorage-independent growth via Rho GDP dissociation inhibitor beta in melanoma B16 cells.

    PubMed

    Wang, Pu; Xu, Su; Wang, Yinan; Wu, Peixing; Zhang, Jinghai; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2011-08-01

    Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells.

  17. A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor.

    PubMed

    Shen, Weitao; Zhang, Xuyao; Fu, Xiang; Fan, Jiajun; Luan, Jingyun; Cao, Zhonglian; Yang, Ping; Xu, Zhongyuan; Ju, Dianwen

    2017-03-30

    Recombinant human arginase (rhArg), an enzyme capable of depleting arginine, has been shown to be an effective therapeutic approach for various cancers. Non-small-cell lung cancer (NSCLC), a histological subtype of pulmonary carcinoma, has a high rate of morbidity and mortality in the world. Thus, the need for novel and more effective treatment is urgent. In this study, it is the first time to report that rhArg could induce significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells. Subsequently, our research revealed that rhArg dramatically stimulated autophagic response in NSCLC cells, which was proved by the formation and accumulation of autophagosomes and the conversion of microtubule-associated protein light chain 3 (LC3) from LC3-I to LC3-II. Furthermore, blocking autophagy by chloroquine or LY294002 remarkably enhanced rhArg-induced cytotoxicity and caspase-dependent apoptosis, suggesting that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells. Further experiments showed that two signaling pathways including the Akt/mTOR and extracellular signal-regulated kinase pathway, and mitochondrial-derived reactive oxygen species (ROS) production were involved in rhArg-induced autophagy and apoptosis. Meanwhile, N-acetyl-L-cysteine, a common antioxidant, was employed to scavenge ROS, and we detected that it could significantly block rhArg-induced autophagy and cytotoxicity, indicating that ROS played a vital role in arginine degradation therapy. Besides, xenograft experiment showed that combination with autophagy inhibitor potentiated the anti-tumor efficacy of rhArg in vivo. Therefore, these results provided a novel prospect and viewpoint that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells, and treatment with rhArg alone or combined with autophagy inhibitor could be a novel and promising therapeutic approach for NSCLC in vivo and in vitro.

  18. Gastrin decreases Na+,K+-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells.

    PubMed

    Liu, Tianbing; Konkalmatt, Prasad R; Yang, Yu; Jose, Pedro A

    2016-04-01

    The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes.

  19. Lipoteichoic acid promotes nuclear accumulation of β-catenin via AKT in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Cardoso-Jiménez, Patricia

    2011-09-01

    Treatment of human gingival fibroblasts (HGFs) with lipoteichoic acid (LTA) results in the activation of multiple signaling pathways. Exposure of HGF to LTA has been shown to result in the activation of phosphatidylinositol 3-kinase (PI3K). The aim of this study was to evaluate the effects of LTA-induced PI3K activation in HGFs. We found that LTA treatment results in the phosphorylation of AKT and glycogen synthase kinase (GSK-3). Inactivation of GSK-3 promotes the nuclear accumulation of β-catenin and expression of connexin43. Treatment with PI3K inhibitors, wortmannin and LY294002, inhibited LTA-induced phosphorylation of AKT and GSK-3, demonstrating that these events require PI3K activation. This report is the first demonstration that LTA treatment activates AKT in HGFs.

  20. Apical phosphatidylserine externalization in auditory hair cells.

    PubMed

    Shi, Xiaorui; Gillespie, Peter G; Nuttall, Alfred L

    2007-01-01

    In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na(+) influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na(+) entry into hair cells.

  1. Molecular cloning and characterization of a threonine/serine protein kinase lvakt from Litopenaeus vannamei

    NASA Astrophysics Data System (ADS)

    Ruan, Lingwei; Liu, Rongdiao; Xu, Xun; Shi, Hong

    2014-07-01

    The phosphatidylinositol 3-kinase (PI3K)-AKT pathway is involved in various cellular functions, including anti-apoptosis, protein synthesis, glucose metabolism and cell cycling. However, the role of the PI3K-AKT pathway in crustaceans remains unclear. In the present study, we cloned and characterized the AKT gene lvakt from Litopenaeus vannamei. The 511-residue LVAKT was highly conserved; contained a PH domain, a catalytic domain and a hydrophobic domain; and was highly expressed in the heart and gills of L. vannamei. We found, using Real-Time Quantitative PCR (Q-PCR) analysis, that lvakt was up-regulated during early white spot syndrome virus (WSSV) infection. Moreover, the PI3K-specific inhibitor, LY294002, reduced viral gene transcription, implying that the PI3K-AKT pathway might be hijacked by WSSV. Our results therefore suggest that LVAKT may play an important role in the shrimp immune response against WSSV.

  2. Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

    PubMed Central

    Baskaran, Sulochanadevi; Carlson, Lars-Anders; Stjepanovic, Goran; Young, Lindsey N; Kim, Do Jin; Grob, Patricia; Stanley, Robin E; Nogales, Eva; Hurley, James H

    2014-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001 PMID:25490155

  3. Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

    PubMed

    Uchida, Yasunori; Rutaganira, Florentine U; Jullié, Damien; Shokat, Kevan M; von Zastrow, Mark

    2017-01-01

    G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.

  4. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  5. PI3 kinase enzymology on fluid lipid bilayers.

    PubMed

    Dutta, Debjit; Pulsipher, Abigail; Luo, Wei; Yousaf, Muhammad N

    2014-10-21

    We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.

  6. Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

    PubMed Central

    McIlroy, J; Chen, D; Wjasow, C; Michaeli, T; Backer, J M

    1997-01-01

    We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway. PMID:8972205

  7. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

  8. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  9. Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

    PubMed

    Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

    2015-10-01

    TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells.

  10. Phosphatidylinositol 3-kinase mediates the ability of retinol to decrease colorectal cancer cell invasion.

    PubMed

    Lengyel, Jennifer N Griffin; Park, Eun Young; Brunson, Anna R; Pinali, Daniel; Lane, Michelle A

    2014-01-01

    Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor-independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

  11. Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage

    PubMed Central

    Angulo, Ivan; Vadas, Oscar; Garçon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R.; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gašper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debré, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A.; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C.; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin; Cant, Andrew J.; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L.; Condliffe, Alison; Nejentsev, Sergey

    2014-01-01

    Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS. PMID:24136356

  12. Phosphoinositide-3-kinases as the novel therapeutic targets for the inflammatory diseases: Current and future perspectives.

    PubMed

    Vyas, Preeti; Vohora, Divya

    2016-10-13

    Recent findings have publicized phosphoinositide-3-kinases (PI3Ks) as novel therapeutic targets, which are also purported to be involved in the complex pathophysiology of inflammatory and various other diseases. They are recognized to participate in the inflammatory cellular responses by modulating the growth, development and proliferation of various immune cells and hence, affect the release of various cytokines and other inflammatory mediators involved in these manifestations. The review presents a brief synopsis of the PI3K/AKT/mTOR signalling pathway along with the current and future prospects of targeting PI3Ks for various diseases, like malignant, autoimmune, inflammatory, cardiovascular, neurological disorders etc., laying special emphasis on the inflammatory diseases and associated cellular responses. The recent literature relating this pathway with these diseases is highlighted, with a hope, which remains for the progression of PI3K inhibitors in the market as a treatment option. With Idelalisib entering the market for cancer, PI3K/AKT signalling has also gained significance as an investigational target for other diseases, particularly for inflammation. Further exploration of this pathway may also uncover its involvement in these disorders, which may further contribute to developing the new treatments and can turn out to be an innovative brainwave in the field of experimental and clinical pharmacology in future.

  13. Tyrosol Suppresses Allergic Inflammation by Inhibiting the Activation of Phosphoinositide 3-Kinase in Mast Cells.

    PubMed

    Je, In-Gyu; Kim, Duk-Sil; Kim, Sung-Wan; Lee, Soyoung; Lee, Hyun-Shik; Park, Eui Kyun; Khang, Dongwoo; Kim, Sang-Hyun

    2015-01-01

    Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of κB kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders.

  14. Phosphoinositide 3-kinase: a new kid on the block in vascular anomalies.

    PubMed

    Castillo, Sandra D; Vanhaesebroeck, Bart; Sebire, Neil J

    2016-12-01

    Vascular anomalies are broadly divided into vascular tumours and malformations. These lesions are composed of abnormal vascular elements of various types, and mainly affect infants, children, and young adults. Vascular anomalies may be painful, may be complicated by bleeding, infection, or organ dysfunction, and can have secondary effects on other tissues. Current treatment strategies include surgical excision, pulsed laser, and sclerotherapy, which are invasive, with risks of recurrence. There are growing pharmacological options for these vascular anomalies, but, to date, no specific targeted therapies have been developed. Phosphoinositide 3-kinases (PI3Ks) constitute a family of lipid kinases that are involved in signal transduction and vesicular traffic, and that modulate important cellular processes such as proliferation, growth, and migration. Recent findings have indicated that the PI3K signalling pathway is important in the pathogenesis of vascular anomalies. This provides an opportunity to use PI3K inhibitors, which are in clinical trials for cancer treatment, for such lesions. Here, we provide an update on the classification of vascular anomalies, with their major features, and discuss the role of the PI3K signalling pathway in the pathogenesis of vascular anomalies, and their clinical implications and therapeutic opportunities. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  15. Phosphoinositide 3-Kinase Gamma Contributes to Neuroinflammation in a Rat Model of Surgical Brain Injury

    PubMed Central

    Huang, Lei; Sherchan, Prativa; Wang, Yuechun; Reis, Cesar; Applegate, Richard L.; Tang, Jiping

    2015-01-01

    Neuroinflammation plays an important role in the pathophysiology of surgical brain injury (SBI). Phosphoinositide 3-kinase gamma (PI3Kγ), predominately expressed in immune and endothelial cells, activates multiple inflammatory responses. In the present study, we investigated the role of PI3Kγ and PI3Kγ-activated phosphodiesterase 3B (PDE3B) in neuroinflammation in a rat model of SBI. One hundred and fifty-two male Sprague Dawley rats (weight 280–350 g) were subjected to a partial right frontal lobe corticotomy model of SBI. A PI3Kγ pharmacological inhibitor (AS252424 or AS605240) was administered intraperitoneally. PI3Kγ siRNA, human recombinant active-PI3Kγ protein, or human recombinant active-PDE3B protein were administered intracerebroventricularly. Post-SBI assessments included neurobehavioral tests, brain water content, Western blot, and immunohistochemistry. Endogenous PI3Kγ levels were increased within peri-resection brain tissues after SBI, accompanied by increased brain water content and neurological functional deficits. There was a trend toward increased endogenous PDE3B phosphorylation after SBI. The selective PI3Kγ inhibitors AS252424 and AS605240 reduced brain water content surrounding corticotomy and improved neurological function after SBI. SBI increased and PI3Kγ inhibitor decreased levels of myeloperoxidase, cluster of differentiation 3, mast cell degranulation, E-selectin, and IL-1 in peri-resection brain tissues. Direct administration of human recombinant active-PI3Kγ protein and active-PDE3B protein countered the protective effect of AS252424. PI3Kγ siRNA reduced PI3Kγ levels, decreased brain water content within peri-resection brain tissues, and improved neurological function after SBI. Collectively, our findings suggest that PI3Kγ contributed to neuroinflammation after SBI. The use of selective PI3Kγ inhibitors may be a novel approach to ameliorating SBI via their anti-inflammation effects. SIGNIFICANCE STATEMENT Life-saving or

  16. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  17. Role of phosphoinositide 3-kinase in the pathogenesis of acute pancreatitis

    PubMed Central

    Lupia, Enrico; Pigozzi, Luca; Goffi, Alberto; Hirsch, Emilio; Montrucchio, Giuseppe

    2014-01-01

    A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity. Thus, research has recently focused on molecules that can regulate the inflammatory processes, such as phosphoinositide 3-kinases (PI3Ks), a family of lipid and protein kinases involved in intracellular signal transduction. Studies using genetic ablation or pharmacologic inhibitors of different PI3K isoforms, in particular the class I PI3Kδ and PI3Kγ, have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses. Recent data suggest that PI3Ks are also involved in the pathogenesis of acute pancreatitis. Activation of the PI3K signaling pathway, and in particular of the class IB PI3Kγ isoform, has a significant role in those events which are necessary for the initiation of acute pancreatic injury, namely calcium signaling alteration, trypsinogen activation, and nuclear factor-κB transcription. Moreover, PI3Kγ is instrumental in modulating acinar cell apoptosis, and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis. The availability of PI3K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease. This article presents a brief summary of PI3K structure and function, and highlights recent advances that implicate PI3Ks in the pathogenesis of acute pancreatitis. PMID:25386068

  18. Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

    PubMed Central

    Higaki, Yasuki; Mikami, Toshio; Fujii, Nobuharu; Hirshman, Michael F.; Koyama, Katsuhiro; Seino, Tetsuya; Tanaka, Keitaro; Goodyear, Laurie J.

    2010-01-01

    We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 μmol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-L-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 μmol/l had no effect on ATP concentrations and did not increase the activities of either the α1 or α2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. PMID:18303121

  19. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  20. Gastrin induces sodium-hydrogen exchanger 3 phosphorylation and mTOR activation via a phosphoinositide 3-kinase-/protein kinase C-dependent but AKT-independent pathway in renal proximal tubule cells derived from a normotensive male human.

    PubMed

    Liu, Tianbing; Jose, Pedro A

    2013-02-01

    Gastrin is natriuretic, but its renal molecular targets and signal transduction pathways are not fully known. In this study, we confirmed the existence of CCKBR (a gastrin receptor) in male human renal proximal tubule cells and discovered that gastrin induced S6 phosphorylation, a downstream component of the phosphatidylinositol 3 kinase (PI3 kinase)-mammalian target of rapamycin pathway. Gastrin also increased the phosphorylation of sodium-hydrogen exchanger 3 (NHE3) at serine 552, caused its internalization, and decreased its expression at the cell surface and NHE activity. The phosphorylation of NHE3 and S6 was dependent on PI3 kinases because it was blocked by 2 different PI3-kinase inhibitors, wortmannin and LY294,002. The phosphorylation of NHE3 and S6 was not affected by the protein kinase A inhibitor H-89 but was blocked by a pan-PKC (chelerythrine) and a conventional PKC (cPKC) inhibitor (Gö6976) (10 μM) and an intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The cPKC involved was probably PKCα because it was phosphorylated by gastrin. The gastrin-mediated phosphorylation of NHE3, S6, and PKCα was via phospholipase C because it was blocked by a phospholipase C inhibitor, U73122 (10 μM). The phosphorylation (activation) of AKT, which is usually upstream of mammalian target of rapamycin in the classic PI3 kinase-AKT-p70S6K signaling pathway, was not affected, suggesting that the gastrin-induced phosphorylation of NHE3 and S6 is dependent on both PI3 kinase and PKCα but not AKT.

  1. Donepezil, an acetylcholine esterase inhibitor, and ABT-239, a histamine H3 receptor antagonist/inverse agonist, require the integrity of brain histamine system to exert biochemical and procognitive effects in the mouse.

    PubMed

    Provensi, Gustavo; Costa, Alessia; Passani, M Beatrice; Blandina, Patrizio

    2016-10-01

    Histaminergic H3 receptors (H3R) antagonists enhance cognition in preclinical models and modulate neurotransmission, in particular acetylcholine (ACh) release in the cortex and hippocampus, two brain areas involved in memory processing. The cognitive deficits seen in aging and Alzheimer's disease have been associated with brain cholinergic deficits. Donepezil is one of the acetylcholinesterase (AChE) inhibitor approved for use across the full spectrum of these cognitive disorders. We addressed the question if H3R antagonists and donepezil require an intact histamine neuronal system to exert their procognitive effects. The effect of the H3R antagonist ABT-239 and donepezil were evaluated in the object recognition test (ORT), and on the level of glycogen synthase kinase 3 beta (GSK-3β) phosphorylation in normal and histamine-depleted mice. Systemic administration of ABT-239 or donepezil ameliorated the cognitive performance in the ORT. However, these compounds were ineffective in either genetically (histidine decarboxylase knock-out, HDC-KO) or pharmacologically, by means of intracerebroventricular (i.c.v.) injections of the HDC irreversible inhibitor a-fluoromethylhistidine (a-FMHis), histamine-deficient mice. Western blot analysis revealed that ABT-239 or donepezil systemic treatments increased GSK-3β phosphorylation in cortical and hippocampal homogenates of normal, but not of histamine-depleted mice. Furthermore, administration of the PI3K inhibitor LY294002 that blocks GSK-3β phosphorylation, prevented the procognitive effects of both drugs in normal mice. Our results indicate that both donepezil and ABT-239 require the integrity of the brain histaminergic system to exert their procognitive effects and strongly suggest that impairments of PI3K/AKT/GSK-3β intracellular pathway activation is responsible for the inefficacy of both drugs in histamine-deficient animals.

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

    PubMed Central

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

    2013-01-01

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

  3. Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

    PubMed Central

    Jeong, Ae-Jin; Chung, Chung-Nam; Kim, Hye-Jin; Bae, Kil Soo; Choi, Song; Jun, Woo Jin; Shim, Sang In; Kang, Tae-Hong; Leem, Sun-Hee

    2012-01-01

    Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway. PMID:23118562

  4. Amino acid availability regulates S6K1 and protein synthesis in avian insulin-insensitive QM7 myoblasts.

    PubMed

    Tesseraud, Sophie; Bigot, Karine; Taouis, Mohammed

    2003-04-10

    The regulation of S6K1 by nutritional status and insulin has been recently reported in vivo in chicken muscle despite the relative insulin resistance of this tissue as estimated by phosphatidylinositol 3-kinase (PI3-kinase) activity. The present work aimed to study the impact of amino acids on S6K1 activity in quail muscle (QM7) myoblasts. Firstly, we characterized S6K1 in QM7 cells and demonstrated the absence of insulin receptors in these cells. Secondly, we showed that amino acids in the absence of insulin induced S6K1 phosphorylation on Thr389 and concomitantly increased its enzymatic activity. Amino acid-induced S6K1 activation was inhibited by LY294002 (PI3-kinase inhibitor) and rapamycin (inhibitor of the mammalian target of rapamycin, mTOR), suggesting the involvement of an avian homolog of mTOR. The availability of individual amino acids (methionine or leucine) regulated S6K1 phosphorylation on Thr389 and QM7 protein synthesis. In conclusion, amino acids regulate S6K1 phosphorylation and activity in QM7 cells through the mTOR/PI3-kinase pathway in an insulin-independent manner.

  5. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    PubMed

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

  6. {alpha}MSH and Cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism.

    PubMed

    Cheli, Yann; Luciani, Flavie; Khaled, Mehdi; Beuret, Laurent; Bille, Karine; Gounon, Pierre; Ortonne, Jean-Paul; Bertolotto, Corine; Ballotti, Robert

    2009-07-10

    Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.

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

    PubMed

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

    2009-12-01

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

  8. Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun N-terminal kinase in paraquat-induced cell death.

    PubMed

    Niso-Santano, Mireia; Morán, José M; García-Rubio, Lourdes; Gómez-Martín, Ana; González-Polo, Rosa A; Soler, Germán; Fuentes, José M

    2006-08-01

    Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.

  9. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  10. Activation of the phosphatidylinositol 3-kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation.

    PubMed

    Wang, Guoliang; Ma, Ning; Meng, Liukun; Wei, Yingjie; Gui, Jingang

    2015-12-01

    Over-activated PI3K/Akt signaling, a pathway strongly related to cancer survival and proliferation, has been reported recently to be involved in pulmonary artery smooth muscle cell apoptosis and proliferation in pulmonary hypertension (PH). In this study, we observed greatly increased lipocalin-2 (Lcn2) expression accompanied with over-activated PI3K/Akt signaling in a standard rat model of PH induced by monocrotaline. In view of the close relationship between Lcn2 and PI3K/Akt pathway, we hypothesized that the up-regulated Lcn2 might be a trigger of over-activated PI3K/Akt signaling in PH. Our results showed that Lcn2 significantly activated the PI3K/Akt pathway (determined by augmented Akt phosphorylation and up-regulated Mdm2) and significantly promoted proliferation (assessed by Ki67 staining) in cultured human pulmonary artery smooth muscle cells. Furthermore, we demonstrated that inhibition of Akt phosphorylation (LY294002) abrogated the Lcn2-promoted proliferation in cultured human pulmonary artery smooth muscle cells. In conclusion, Lcn2 significantly promoted human pulmonary artery smooth muscle cell proliferation by activating PI3K/Akt pathway. Further study on the role and mechanism of Lcn2 will help explore novel therapeutic strategies based on attenuating over-activated PI3K/Akt signaling in PH.

  11. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression

    PubMed Central

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-01-01

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis. PMID:27071537

  12. 2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061), a novel microtubule inhibitor, evokes G2/M cell cycle arrest and apoptosis in human breast cancer cells.

    PubMed

    Xu, Jingwen; Zuo, Daiying; Qi, Huan; Shen, Qirong; Bai, Zhaoshi; Han, Mengting; Li, Zengqiang; Zhang, Weige; Wu, Yingliang

    2016-03-01

    Breast cancer is the leading cause of cancer death in women worldwide, and novel chemotherapeutic drugs with high activity and no drug resistance for treating breast cancer are needed urgently. In this study, we investigated the antitumor effect of 2-methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061), which has a strong inhibition of cell growth in MCF-7 and MDA-MB-231 cells. We demonstrated that SQ0814061 (SQ) time-dependently induced cell cycle arrest at G2/M phase and subsequently progressed into apoptosis, which is associated with microtubule depolymerization. Western blot analysis revealed that up-regulation of cyclin B1 and Aurora A was related with G2/M phase arrest in MCF-7 and MDA-MB-231 cells treatment with SQ. However, the formation of multinucleated cells after a long time exposed to SQ of MCF-7 cells delayed the cell death. In addition, apoptosis induced by SQ is correlated with the down-regulation of the PI3K-Akt-MDM2 pathway in MCF-7 and MDA-MB-231 cells. Treatment with the PI3K specific inhibitor, LY294002, increased SQ-induced cell growth inhibitory rate and apoptosis rate of MCF-7 and MDA-MB-231 cells. Moreover, SQ induced MCF-7 and MDA-MB-231 cells to generate reactive oxygen species (ROS), and the SQ-induced cell death was ROS dependent. In conclusion, all the data demonstrated that SQ exhibited its antitumor activity through disrupting the microtubule assembly, inducing cell cycle arrest and eventually apoptosis which is associated with PI3K-Akt-MDM2 pathway in MCF-7 and MDA-MB-231 cells. Therefore, the novel compound SQ is a promising microtubule inhibitor that has tremendous potentials for therapeutic treatment of human mastocarcinoma.

  13. Expression and regulation of endothelial nitric oxide synthase by vascular endothelial growth factor in ECV 304 cells.

    PubMed Central

    Park, Jong Seon; Hong, Gu Ru; Baek, Suk Whan; Shin, Dong Gu; Kim, Young Jo; Shim, Bong Sup

    2002-01-01

    Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with VEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to VEGF(165), eNOS activity and cell growth were increased by approximately two-fold in the VEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, VEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway. PMID:11961297

  14. Factors affecting directional migration of bone marrow mesenchymal stem cells to the injured spinal cord.

    PubMed

    Xia, Peng; Pan, Su; Cheng, Jieping; Yang, Maoguang; Qi, Zhiping; Hou, Tingting; Yang, Xiaoyu

    2014-09-15

    Microtubule-associated protein 1B plays an important role in axon guidance and neuronal migration. In the present study, we sought to discover the mechanisms underlying microtubule-associated protein 1B mediation of axon guidance and neuronal migration. We exposed bone marrow mesenchymal stem cells to okadaic acid or N-acetyl-D-erythro-sphingosine (an inhibitor and stimulator, respectively, of protein phosphatase 2A) for 24 hours. The expression of the phosphorylated form of type I microtubule-associated protein 1B in the cells was greater after exposure to okadaic acid and lower after N-acetyl-D-erythro-sphingosine. We then injected the bone marrow mesenchymal stem cells through the ear vein into rabbit models of spinal cord contusion. The migration of bone marrow mesenchymal stem cells towards the injured spinal cord was poorer in cells exposed to okadaic acid- and N-acetyl-D-erythro-sphingosine than in non-treated bone marrow mesenchymal stem cells. Finally, we blocked phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways in rabbit bone marrow mesenchymal stem cells using the inhibitors LY294002 and U0126, respectively. LY294002 resulted in an elevated expression of phosphorylated type I microtubule-associated protein 1B, whereas U0126 caused a reduction in expression. The present data indicate that PI3K and ERK1/2 in bone marrow mesenchymal stem cells modulate the phosphorylation of microtubule-associated protein 1B via a cross-signaling network, and affect the migratory efficiency of bone marrow mesenchymal stem cells towards injured spinal cord.

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

    SciTech Connect

    Zhang, Yuqin; Zheng, Lin; Ding, Yi; Li, Qi; Wang, Rong; Liu, Tongxin; Sun, Quanquan; Yang, Hua; Peng, Shunli; Wang, Wei; Chen, Longhua

    2015-08-01

    Purpose: To investigate the role of miR-20a in hepatocellular carcinoma (HCC) cell radioresistance, which may reveal potential strategies to improve treatment. Methods and Materials: The expression of miR-20a and PTEN were detected in HCC cell lines and paired primary tissues by quantitative real-time polymerase chain reaction. Cell radiation combined with colony formation assays was administrated to discover the effect of miR-20a on radiosensitivity. Bioinformatics prediction and luciferase assay were used to identify the target of miR-20a. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit phosphorylation of Akt, to verify whether miR-20a affects HCC cell radioresistance through activating the PTEN/PI3K/Akt pathway. Results: MiR-20a levels were increased in HCC cell lines and tissues, whereas PTEN was inversely correlated with it. Overexpression of miR-20a in Bel-7402 and SMMC-7721 cells enhances their resistance to the effect of ionizing radiation, and the inhibition of miR-20a in HCCLM3 and QGY-7701 cells sensitizes them to it. PTEN was identified as a direct functional target of miR-20a for the induction of radioresistance. Overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway. Additionally, the kinase inhibitor LY294002 could reverse the effect of miR-20a–induced radioresistance. Conclusion: MiR-20a induces HCC cell radioresistance by activating the PTEN/PI3K/Akt pathway, which suggests that miR-20a/PTEN/PI3K/Akt might represent a target of investigation for developing effective therapeutic strategies against HCC.

  16. Melatonin attenuates sepsis-induced cardiac dysfunction via a PI3K/Akt-dependent mechanism.

    PubMed

    An, Rui; Zhao, Lei; Xi, Cong; Li, Haixun; Shen, Guohong; Liu, Haixiao; Zhang, Shumiao; Sun, Lijun

    2016-01-01

    Myocardial dysfunction is an important manifestation of sepsis. Previous studies suggest that melatonin is protective against sepsis. In addition, activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway has been reported to be beneficial in sepsis. However, the role of PI3K/Akt signaling in the protective effect of melatonin against sepsis-induced myocardial dysfunction remains unclear. Here, LY294002, a PI3K inhibitor, was used to investigate the role of PI3K/Akt signaling in mediating the effects of melatonin on sepsis-induced myocardial injury. Cecal ligation and puncture (CLP) surgery was used to establish a rat model of sepsis. Melatonin was administrated to rats intraperitoneally (30 mg/kg). The survival rate, measures of myocardial injury and cardiac performance, serum lactate dehydrogenase level, inflammatory cytokine levels, oxidative stress level, and the extent of myocardial apoptosis were assessed. The results suggest that melatonin administration after CLP surgery improved survival rates and cardiac function, attenuated myocardial injury and apoptosis, and decreased the serum lactate dehydrogenase level. Melatonin decreased the production of the inflammatory cytokines TNF-α, IL-1β, and HMGB1, increased anti-oxidant enzyme activity, and decreased the expression of markers of oxidative damage. Levels of phosphorylated Akt (p-Akt), unphosphorylated Akt (Akt), Bcl-2, and Bax were measured by Western blot. Melatonin increased p-Akt levels, which suggests Akt pathway activation. Melatonin induced higher Bcl-2 expression and lower Bax expression, suggesting inhibition of apoptosis. All protective effects of melatonin were abolished by LY294002, the PI3K inhibitor. In conclusion, our results demonstrate that melatonin mitigates myocardial injury in sepsis via PI3K/Akt signaling activation.

  17. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  18. Berberine protects endothelial progenitor cell from damage of TNF-α via the PI3K/AKT/eNOS signaling pathway.

    PubMed

    Xiao, Min; Men, Li Na; Xu, Ming Guo; Wang, Guo Bing; Lv, Hai Tao; Liu, Cong

    2014-11-15

    Endothelial progenitor cells (EPCs) dysfunction is closely correlated with the coronary artery injury induced by Kawasaki disease (KD). The level of tumor necrosis factor-α (TNF-α) elevated significantly in acute phase of KD which can damage the functions of EPCs. The aim of this study was to investigate whether berberine (BBR) can protect EPCs from the inhibition caused by TNF-α via the PI3K (Phosphatidyl Inositol 3-kinase) /AKT (Serine/threonine protein kinase B) /eNOS (endothelial Nitric Oxide synthase) signaling pathway. The cell proliferative ability of EPCs was determined by MTT (methyl thiazolyl tetrazolium) assays. Nitric oxide (NO) level was determined in supernatants. The mRNA level of eNOS, PI3K and AKT were measured by Real Time-Polymerase Chain Reaction (RT-PCR), and the protein levels of eNOS, phospho-eNOS (p-eNOS), Akt, phospho-Akt (p-Akt) and PI3K were analyzed using Western-blot. The results demonstrated that TNF-α inhibits the proliferative ability of EPCs. However, BBR improves the proliferative activity of EPCs inhibited by TNF-α. Blockade of PI3K by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) and blockade of eNOS by l-NAME (NG-Nitroarginine Methyl Ester) attenuates the effect of BBR. BBR can increase the level of PI3K/Akt/eNOS mRNA and the protein level of PI3K, p-Akt, eNOS and p-eNOS, which can be blocked by PI3K inhibitor (LY294002) and eNOS inhibitor (l-NAME). Therefore, we concluded that impaired EPCs proliferation could be reversed by BBR via the PI3K/AKT/eNOS signaling pathway.

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

    PubMed Central

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

    2012-01-01

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

  20. Interleukin-13-induced MUC5AC expression is regulated by a PI3K–NFAT3 pathway in mouse tracheal epithelial cells

    SciTech Connect

    Yan, Fugui; Li, Wen; Zhou, Hongbin; Wu, Yinfang; Ying, Songmin; Chen, Zhihua; Shen, Huahao

    2014-03-28

    Highlights: • IL-13 specifically induced NFAT3 activation in mouse tracheal epithelial cells. • CsA and LY294002 significantly blocked IL-13-induced MUC5AC production. • The PI3K–NFAT3 pathway is positively involved in IL-13-induced MUC5AC production. - Abstract: Interleukin-13 (IL-13) plays a critical role in asthma mucus overproduction, while the mechanisms underlying this process are not fully elucidated. Previous studies showed that nuclear factor of activated T cells (NFAT) is involved in the pathogenesis of asthma, but whether it can directly regulate IL-13-induced mucus (particularly MUC5AC) production is still not clear. Here we showed that IL-13 specifically induced NFAT3 activation through promoting its dephosphorylation in air–liquid interface (ALI) cultures of mouse tracheal epithelial cells (mTECs). Furthermore, both Cyclosporin A (CsA, a specific NFAT inhibitor) and LY294002 (a Phosphoinositide 3-kinase (PI3K) inhibitor) significantly blocked IL-13-induced MUC5AC mRNA and protein production through the inhibition of NFAT3 activity. We also confirmed that CsA could not influence the forkhead Box A2 (Foxa2) and mouse calcium dependent chloride channel 3 (mClca3) expression in IL-13-induced MUC5AC production, which both are known to be important in IL-13-stimulated mucus expression. Our study is the first to demonstrate that the PI3K–NFAT3 pathway is positively involved in IL-13-induced mucus production, and provided novel insights into the molecular mechanism of asthma mucus hypersecretion.

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

    SciTech Connect

    Lin, W.-N.; Luo, S.-F.; Wu, C.-B.; Lin, C.-C.; Yang, C.-M.

    2008-04-15

    In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-{kappa}B 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.

  2. Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells.

    PubMed

    Shin, Sooan; Paul-Satyaseela, Maneesh; Maneesh, Paul-Satyaseela; Lee, Jong-Seok; Romer, Lewis H; Kim, Kwang Sik

    2006-01-01

    Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.

  3. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

  4. Multiple roles for PI 3-kinase in the regulation of PLCgamma activity and Ca2+ mobilization in antigen-stimulated mast cells.

    PubMed

    Barker, S A; Lujan, D; Wilson, B S

    1999-03-01

    Cross-linking the IgE-bound FcepsilonRI with polyvalent antigen leads to Ca2+-dependent degranulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of FcepsilonRI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLCgamma activity and is demonstrated by a dramatic inhibition of FcepsilonRI-induced Ins(1,4,5)P3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P3 production in at least two ways: by promoting translocation and phosphorylation of PLCgamma1 and by direct stimulation of both PLCgamma isoforms. In vitro stimulation of PLCgamma activity by PtdIns(3,4,5)P3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compared with control cells, Ca2+ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca2+ after antigen and an inhibition of the sustained Ca2+ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca2+ stores in antigen-stimulated cells in the presence or absence of wortmannin. These data suggest that sufficient Ins(1,4,5)P3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.

  5. Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

    PubMed Central

    Williams, Karen D.; Busto, Macarena; Suster, Maximiliano L.; So, Anthony K.-C.; Ben-Shahar, Yehuda; Leevers, Sally J.; Sokolowski, Marla B.

    2006-01-01

    This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses. PMID:17043223

  6. Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

    PubMed Central

    Crljen, Vladiana; Volinia, Stefano; Banfic, Hrvoje

    2002-01-01

    Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and

  7. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration.

    PubMed

    Dise, Rebecca S; Frey, Mark R; Whitehead, Robert H; Polk, D Brent

    2008-01-01

    Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

  8. Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

    PubMed Central

    Bruel-Jungerman, Elodie; Veyrac, Alexandra; Dufour, Franck; Horwood, Jennifer; Laroche, Serge; Davis, Sabrina

    2009-01-01

    Background Physical exercise has been shown to increase adult neurogenesis in the dentate gyrus and enhances synaptic plasticity. The antiapoptotic kinase, Akt has also been shown to be phosphorylated following voluntary exercise; however, it remains unknown whether the PI3K-Akt signaling pathway is involved in exercise-induced neurogenesis and the associated facilitation of synaptic plasticity in the dentate gyrus. Methodology/Principal Findings To gain insight into the potential role of this signaling pathway in exercise-induced neurogenesis and LTP in the dentate gyrus rats were infused with the PI3K inhibitor, LY294002 or vehicle control solution (icv) via osmotic minipumps and exercised in a running wheel for 10 days. Newborn cells in the dentate gyrus were date-labelled with BrdU on the last 3 days of exercise. Then, they were either returned to the home cage for 2 weeks to assess exercise-induced LTP and neurogenesis in the dentate gyrus, or were killed on the last day of exercise to assess proliferation and activation of the PI3K-Akt cascade using western blotting. Conclusions/Significance Exercise increases cell proliferation and promotes survival of adult-born neurons in the dentate gyrus. Immediately after exercise, we found that Akt and three downstream targets, BAD, GSK3β and FOXO1 were activated. LY294002 blocked exercise-induced phosphorylation of Akt and downstream target proteins. This had no effect on exercise-induced cell proliferation, but it abolished most of the beneficial effect of exercise on the survival of newly generated dentate gyrus neurons and prevented exercise-induced increase in dentate gyrus LTP. These results suggest that activation of the PI3 kinase-Akt signaling pathway plays a significant role via an antiapoptotic function in promoting survival of newly formed granule cells generated during exercise and the associated increase in synaptic plasticity in the dentate gyrus. PMID:19936256

  9. Blood -brain barrier disruption was less under isoflurane than pentobarbital anesthesia via a PI3K/Akt pathway in early cerebral ischemia.

    PubMed

    Chi, Oak Z; Mellender, Scott J; Kiss, Geza K; Liu, Xia; Weiss, Harvey R

    2017-02-24

    One of the important factors altering the degree of blood-brain barrier (BBB) disruption in cerebral ischemia is the anesthetic used. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been reported to be involved in modulating BBB permeability and in isoflurane induced neuroprotection. This study was performed to compare the degree of BBB disruption in focal cerebral ischemia under isoflurane vs pentobarbital anesthesia and to determine whether inhibition of PI3K/Akt would affect the disruption in the early stage of focal cerebral ischemia. Permanent middle cerebral artery (MCA) occlusion was performed in rats under 1.4% isoflurane or pentobarbital (50mg/kg i.p.) anesthesia with controlled ventilation. In half of each group LY294002, which is a PI3K/Akt inhibitor, was applied on the ischemic cortex immediately after MCA occlusion. After one hour of MCA occlusion, the transfer coefficient (Ki) of (14)C-α-aminoisobutyric acid ((14)C-AIB) was determined to quantify the degree of BBB disruption. MCA occlusion increased the Ki both in the isoflurane and pentobarbital anesthetized rats. However, the value of Ki was lower under isoflurane (11.5±6.0μL/g/min) than under pentobarbital (18.3±7.1μL/g/min) anesthesia. The Ki of the contralateral cortex of the pentobarbital group was higher (+74%) than that of the isoflurane group. Application of LY294002 on the ischemic cortex increased the Ki (+99%) only in the isoflurane group. The degree of BBB disruption by MCA occlusion was significantly lower under isoflurane than pentobarbital anesthesia in the early stage of cerebral ischemia. Our data demonstrated the importance of choice of anesthetics and suggest that PI3K/Akt signaling pathway plays a significant role in altering BBB disruption in cerebral ischemia during isoflurane but not during pentobarbital anesthesia.

  10. Gefitinib induces lung cancer cell autophagy and apoptosis via blockade of the PI3K/AKT/mTOR pathway

    PubMed Central

    ZHAO, ZHONG-QUAN; YU, ZHONG-YANG; LI, JIE; OUYANG, XUE-NONG

    2016-01-01

    Gefitinib is a selective inhibitor of the tyrosine kinase epidermal growth factor receptor, which inhibits tumor pathogenesis, metastasis and angiogenesis, as well as promoting apoptosis. Therefore, gefitinib presents an effective drug for the targeted therapy of lung cancer. However, the underlying mechanisms by which gefitinib induces lung cancer cell death remain unclear. To investigate the effects of gefitinib on lung cancer cells and the mechanism of such, the present study analyzed the effect of gefitinib on the autophagy, apoptosis and proliferation of the A549 and A549-gefitinib-resistant (GR) cell lines GR. The regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway was also investigated. Acridine orange staining revealed that gefitinib induced autophagy of A549 cells but not A549-GR cells. In addition, gefitinib promoted apoptosis and inhibited proliferation of A549 cells but not A549-GR cells. Furthermore, western blot analysis demonstrated that gefitinib treatment led to the downregulation of PI3K, AKT, pAKT, mTOR and phosphorylated-mTOR protein expression in A549 cells but not A549-GR cells. LY294002 blocked the PI3K/AKT/mTOR pathway and induced autophagy and apoptosis of A549 cells, however, no synergistic effect was observed following combined treatment with gefitinib and LY294002. In conclusion, the results of the present study indicate that gefitinib promotes autophagy and apoptosis of lung cancer cells via blockade of the PI3K/AKT/mTOR pathway, which leads to lung cancer cell death. PMID:27347100

  11. L-F001, a Multifunction ROCK Inhibitor Prevents 6-OHDA Induced Cell Death Through Activating Akt/GSK-3beta and Nrf2/HO-1 Signaling Pathway in PC12 Cells and Attenuates MPTP-Induced Dopamine Neuron Toxicity in Mice.

    PubMed

    Luo, Liting; Chen, Jingkao; Su, Dan; Chen, Meihui; Luo, Bingling; Pi, Rongbiao; Wang, Lan; Shen, Wei; Wang, Rikang

    2017-02-01

    Amounting evidences demonstrated that Rho/Rho-associated kinase (ROCK) might be a novel target for the therapy of Parkinson's disease (PD). Recently, we synthesized L-F001 and revealed it was a potent ROCK inhibitor with multifunctional effects. Here we investigated the effects of L-F001 in PD models. We found that L-F001 potently attenuated 6-OHDA-induced cytotoxicity in PC12 cells and significantly decreased intracellular reactive oxygen species (ROS), prevented the 6-OHDA-induced decline of mitochondrial membrane potential and intracellular GSH levels. In addition, L-F001 increased Akt and GSK-3beta phosphorylation and induced the nuclear Nrf2 and HO-1 expression in a time- and concentration-dependent manner. Moreover, L-F001 restored the levels of p-Akt and p-GSK-3beta (Ser9) as well as HO-1 expression reduced by 6-OHDA. Those effects were blocked by the specific PI3K inhibitor, LY294002, indicating the involvement of Akt/GSK-3beta pathway in the neuroprotective effect of L-F001. In addition, L-F001 significantly attenuated the tyrosinehydroxylase immunoreactive cell loss in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-induced mice PD model. Together, our findings suggest that L-F001 prevents 6-OHDA-induced cell death through activating Akt/GSK-3beta and Nrf2/HO-1 signaling pathway and attenuates MPTP-induced dopaminergic neuron toxicity in mice. L-F001 might be a promising drug candidate for PD.

  12. Erythropoietin protects cardiac myocytes against anthracycline-induced apoptosis

    SciTech Connect

    Fu Ping; Arcasoy, Murat O. . E-mail: arcas001@mc.duke.edu

    2007-03-09

    The cardiotoxic adverse effects of anthracycline antibiotics limit their therapeutic utility as essential components of chemotherapy regimens for hematologic and solid malignancies. Here we show that the hematopoietic cytokine erythropoietin attenuates doxorubicin-induced apoptosis of primary neonatal rat ventricular cardiomyocytes in a dose-dependent manner. Erythropoietin treatment induced rapid, time-dependent phosphorylation of MAP kinases (MAPK) Erk1/2 and the phosphatidylinositol 3-kinase substrate Akt. Treatment of cardiomyocytes with inhibitors of phosphatidylinositol 3-kinase (LY294002) or Akt (Akti-1/2) abolished the protective effect of erythropoietin, whereas treatment with MAPK kinase (MEK1) inhibitor U0126 did not. Erythropoietin also induced the phosphorylation of GSK-3{beta}, a downstream target of PI3K-Akt. Because phosphorylation is known to inactivate GSK-3{beta}, we investigated whether GSK-3{beta} inhibition is cardioprotective. We found that GSK-3{beta} inhibitors SB216763 or lithium chloride blocked doxorubicin-induced cardiomyocyte apoptosis in a manner similar to erythropoietin, suggesting that GSK-3{beta} inhibition is involved in erythropoietin-mediated cardioprotection. Erythropoietin may serve as a novel cardioprotective agent against anthracycline-induced cardiotoxicity.

  13. Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Hwang, Jinah; Sykes, Michelle; Michell, Belinda J.; Kemp, Bruce E.; Lum, Hazel; Jo, Hanjoong

    2002-01-01

    Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.

  14. Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

    PubMed

    Martelli, A M; Evangelisti, C; Follo, M Y; Ramazzotti, G; Fini, M; Giardino, R; Manzoli, L; McCubrey, J A; Cocco, L

    2011-01-01

    Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

  15. Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

    PubMed Central

    Yang, Yanan; Iwanaga, Kentaro; Raso, Maria Gabriela; Wislez, Marie; Hanna, Amy E.; Wieder, Eric D.; Molldrem, Jeffrey J.; Wistuba, Ignacio I.; Powis, Garth; Demayo, Francesco J.; Kim, Carla F.; Kurie, Jonathan M.

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients. PMID:18493606

  16. Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis.

    PubMed

    Mishima, Elina; Sharma, Ashu

    2011-08-01

    Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of 'lipid rafts'), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process.

  17. Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

    PubMed Central

    ZHANG, Zong-Kang; LI, Jie; LIU, Jin; GUO, Baosheng; LEUNG, Albert; ZHANG, Ge; ZHANG, Bao-Ting

    2016-01-01

    Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment could significantly attenuate the decreases in the phosphorylation level of PI3K at p110 catalytic subunit and its downstream markers related to protein synthesis, and inhibit the increases in protein degradation markers at mRNA and protein levels in rat soleus muscle following 28-day hindlimb unloading. In addition, the decreases in soleus muscle mass, muscle fiber cross-sectional area, twitch force, specific force, contraction time and half relaxation time could be significantly attenuated by the high dose ICT treatment. The low dose ICT treatment could moderately attenuate the above changes induced by unloading. Wortmannin, a specific inhibitor of PI3K at p110 catalytic subunit, could abolish the above effects of ICT in vitro and in vivo, indicating that PI3K/Akt signaling could be required by ICT to counteract skeletal muscle atrophy following mechanical unloading. PMID:26831566

  18. Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci*

    PubMed Central

    Popkie, Anthony P.; Zeidner, Leigh C.; Albrecht, Ashley M.; D'Ippolito, Anthony; Eckardt, Sigrid; Newsom, David E.; Groden, Joanna; Doble, Bradley W.; Aronow, Bruce; McLaughlin, K. John; White, Peter; Phiel, Christopher J.

    2010-01-01

    Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. PMID:21047779

  19. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  20. Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes.

    PubMed

    Harris, Stephanie J; Parry, Richard V; Westwick, John; Ward, Stephen G

    2008-02-01

    The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.

  1. The association of phosphoinositide 3-kinase enhancer A with hepatic insulin receptor enhances its kinase activity.

    PubMed

    Chan, Chi Bun; Liu, Xia; He, Kunyan; Qi, Qi; Jung, Dae Y; Kim, Jason K; Ye, Keqiang

    2011-07-01

    Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

  2. Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion

    PubMed Central

    Xie, Yan; Abel, Peter W.; Kirui, Joseph K.; Deng, Caishu; Sharma, Poonam; Wolff, Dennis W.; Toews, Myron L.; Tu, Yaping

    2013-01-01

    Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, β and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kβ. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis. PMID:23500535

  3. Artesunate Protected Blood-Brain Barrier via Sphingosine 1 Phosphate Receptor 1/Phosphatidylinositol 3 Kinase Pathway After Subarachnoid Hemorrhage in Rats.

    PubMed

    Zuo, Shilun; Ge, Hongfei; Li, Qiang; Zhang, Xuan; Hu, Rong; Hu, Shengli; Liu, Xin; Zhang, John H; Chen, Yujie; Feng, Hua

    2017-03-01

    Blood-brain barrier preservation plays an important role in attenuating vasogenic brain edema after subarachnoid hemorrhage (SAH). This study was designed to investigate the protective effect and mechanism of artesunate, a traditional anti-malaria drug, on blood-brain barrier after SAH. Three hundred and seventy-seven (377) male Sprague-Dawley rats were subjected to endovascular perforation model for SAH. The rats received artesunate alone or in combination with Sphingosine-1-phosphate receptor-1 (S1P1) small interfering RNA (siRNA), antagonist VPC23019, or phosphatidylinositol 3-kinase inhibitor wortmannin after SAH. Modified Garcia score, SAH grades, brain water content, Evans blue leakage, transmission electron microscope, immunohistochemistry staining, Western blot, and cultured endothelial cells were used to investigate the optimum concentration and the therapeutic mechanism of artesunate. We found that artesunate (200 mg/kg) could do better in raising modified Garcia score, reducing brain water content and Evans blue leakage than other groups after SAH. Moreover, artesunate elevated S1P1 expression, enhanced phosphatidylinositol 3-kinase activation, lowered GSK-3β activation, stabilized β-catenin, and improved the expression of Claudin-3 and Claudin-5 after SAH in rats. These effects were eliminated by S1P1 siRNA, VPC23019, and wortmannin. This study revealed that artesunate could preserve blood-brain barrier integrity and improve neurological outcome after SAH, possibly through activating S1P1, enhancing phosphatidylinositol 3-kinase activation, stabilizing β-catenin via GSK-3β inhibition, and then effectively raising the expression of Claudin-3 and Claudin-5. Therefore, artesunate may be favorable for the blood-brain barrier (BBB) protection after SAH and become a potential candidate for the treatment of SAH patients.

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

    PubMed Central

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

    2016-01-01

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

  5. Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.

    PubMed

    Matsunaga, Kohichi; Morita, Eiji; Saitoh, Tatsuya; Akira, Shizuo; Ktistakis, Nicholas T; Izumi, Tetsuro; Noda, Takeshi; Yoshimori, Tamotsu

    2010-08-23

    Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

  6. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

  7. Dehydroepiandrosterone Stimulates Phosphorylation of FoxO1 in Vascular Endothelial Cells via Phosphatidylinositol 3-Kinase- and Protein Kinase A-dependent Signaling Pathways to Regulate ET-1 Synthesis and Secretion*

    PubMed Central

    Chen, Hui; Lin, Alice Seraphina; Li, Yunhua; Reiter, Chad E. N.; Ver, Maria R.; Quon, Michael J.

    2008-01-01

    Dehydroepiandrosterone (DHEA) is an endogenous adrenal steroid hormone with controversial actions in humans. We previously reported that DHEA has opposing actions in endothelial cells to stimulate phosphatidylinositol (PI) 3-kinase/Akt/endothelial nitric-oxide synthase leading to increased production of nitric oxide while simultaneously stimulating MAPK-dependent secretion of the vasoconstrictor ET-1. In the present study we hypothesized that DHEA may stimulate PI 3-kinase-dependent phosphorylation of FoxO1 in endothelial cells to help regulate endothelial function. In bovine or human aortic endothelial cells (BAEC and HAEC), treatment with DHEA (100 nm) acutely enhanced phosphorylation of FoxO1. DHEA-stimulated phosphorylation of FoxO1 was inhibited by pretreatment of cells with wortmannin (PI 3-kinase inhibitor) or H89 (protein kinase A (PKA) inhibitor) but not ICI182780 (estrogen receptor blocker), or PD98059 (MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor). Small interfering RNA knockdown of PKA inhibited DHEA-stimulated phosphorylation of FoxO1. DHEA promoted nuclear exclusion of FoxO1 that was blocked by pretreatment of cells with wortmannin, H89, or by small interfering RNA knockdown of PKA. DHEA treatment of endothelial cells increased PKA activity and intracellular cAMP concentrations. Transfection of BAEC with a constitutively nuclear FoxO1 mutant transactivated a co-transfected ET-1 promoter luciferase reporter. Treatment of BAEC with DHEA inhibited transactivation of the ET-1 promoter reporter in cells overexpressing FoxO1. ET-1 promoter activity and secretion in response to DHEA treatment was augmented by PI 3-kinase blockade and inhibited by MAPK blockade. We conclude that DHEA stimulates phosphorylation of FoxO1 via PI 3-kinase- and PKA-dependent pathways in endothelial cells that negatively regulates ET-1 promoter activity and secretion. Balance between PI 3-kinase-dependent inhibition and MAPK-dependent stimulation of ET-1

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

    PubMed

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

    2014-10-01

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

  9. Phosphoinositide-3-kinases p110α and p110β mediate S phase entry in astroglial cells in the marginal zone of rat neocortex.

    PubMed

    Müller, Rabea; Fischer, Catharina; Wilmes, Thomas; Heimrich, Bernd; Distel, Vanessa; Klugbauer, Norbert; Meyer, Dieter K

    2013-01-01

    In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative (dn) isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine (BrdU). Only in astroglial cells harvested from the marginal zone (MZ) of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with BrdU, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110α and p110β were essential for S phase entry. p110α diminished the level of the p27(Kip1) which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110β phosphorylated and inhibited glycogen synthase kinase-3β which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

  10. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    PubMed

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  11. Inhibition of neointimal formation by trans-resveratrol: role of phosphatidyl inositol 3-kinase-dependent Nrf2 activation in heme oxygenase-1 induction.

    PubMed

    Kim, Jung Woo; Lim, Sung Chul; Lee, Moo Yeol; Lee, Jeong Woon; Oh, Won Keun; Kim, Sang Kyum; Kang, Keon Wook

    2010-10-01

    Neointima, defined as abnormal growth of the intimal layer of blood vessels, is believed to be a critical event in the development of vascular occlusive disease. Although resveratrol's inhibitory effects on proliferation and migration of vascular smooth muscle cells has been reported, its activity on neointimal formation is still unclear. Oral administration of trans-resveratrol significantly suppressed intimal hyperplasia in a wire-injured femoral artery mouse model. In cultured vascular smooth muscle cells, trans-resveratrol inhibited platelet-derived growth factor-stimulated DNA synthesis and cell proliferation with down-regulation of cyclin D and pRB. Moreover, platelet-derived growth factor-induced production of reactive oxygen species was inhibited by trans-resveratrol and the compound induced heme oxygenase-1 (HO-1). The anti-proliferative activity of trans-resveratrol was reversed by an HO-1 inhibitor, ZnPPIX. Subcellular fractionation and reporter gene analyses revealed that trans-resveratrol increased the level of nuclear Nrf2 and antioxidant response element reporter activity, and that these were essential for the induction of HO-1. Trans-resveratrol also enhanced the activities of phosphatidyl inositol 3-kinase and extracellular signal regulated kinase, and phosphatidyl inositol 3-kinase was required for Nrf2/antioxidant response element-dependent HO-1 induction. These data have significant implications for the elucidation of the pharmacological mechanism by which trans-resveratrol prevents vascular occlusive diseases.

  12. Deciphering Combinations of PI3K/AKT/mTOR Pathway Drugs Augmenting Anti-Angiogenic Efficacy In Vivo

    PubMed Central

    Sasore, Temitope; Kennedy, Breandán

    2014-01-01

    Ocular neovascularization is a common pathology associated with human eye diseases e.g. age-related macular degeneration and proliferative diabetic retinopathy. Blindness represents one of the most feared disabilities and remains a major burden to health-care systems. Current approaches to treat ocular neovascularisation include laser photocoagulation, photodynamic therapy and anti-VEGF therapies: Ranibizumab (Lucentis) and Aflibercept (Eylea). However, high clinical costs, frequent intraocular injections, and increased risk of infections are challenges related with these standards of care. Thus, there is a clinical need to develop more effective drugs that overcome these challenges. Here, we focus on an alternative approach by quantifying the in vivo anti-angiogenic efficacy of combinations of phosphatidylinositol-3-kinase (PI3K) pathway inhibitors. The PI3K/AKT/mTOR pathway is a complex signalling pathway involved in crucial cellular functions such as cell proliferation, migration and angiogenesis. RT-PCR confirms the expression of PI3K target genes (pik3ca, pik3r1, mtor and akt1) in zebrafish trunks from 6 hours post fertilisation (hpf) and in eyes from 2 days post fertilisation (dpf). Using both the zebrafish intersegmental vessel and hyaloid vessel assays to measure the in vivo anti-angiogenic efficacy of PI3K/Akt/mTOR pathway inhibitors, we identified 5 µM combinations of i) NVP-BEZ235 (dual PI3K-mTOR inhibitor) + PI-103 (dual PI3K-mTOR inhibitor); or ii) LY-294002 (pan-PI3K inhibitor) + NVP-BEZ235; or iii) NVP-BEZ235 + rapamycin (mTOR inhibitor); or iv) LY-294002 + rapamycin as the most anti-angiogenic. Treatment of developing larvae from 2–5 dpf with 5 µM NVP-BEZ235 plus PI-103 resulted in an essentially intact ocular morphology and visual behaviour, whereas other combinations severely disrupted the developing retinal morphology and visual function. In human ARPE19 retinal pigment epithelium cells, however, no significant difference in cell number was

  13. Synthesis and Pharmacological Evaluation of 4-Iminothiazolidinones for Inhibition of PI3 Kinase

    PubMed Central

    Pinson, Jo-Anne; Schmidt-Kittler, Oleg; Frazzetto, Mark; Zheng, Zhaohua; Jennings, Ian G.; Kinzler, Kenneth W.; Vogelstein, Bert; Chalmers, David K.; Thompson, Philip E.

    2012-01-01

    The thiazolidinedione, compound 1, has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound 1 could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity. PMID:23997244

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

    PubMed

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

    2016-12-20

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

  15. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  16. Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1

    PubMed Central

    Zgheel, Faraj; Alhosin, Mahmoud; Rashid, Sherzad; Burban, Mélanie; Auger, Cyril; Schini-Kerth, Valérie B.

    2014-01-01

    Aims Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation. Methods and Results EPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively. Conclusion Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS. PMID:25133540

  17. Thrombopoietin enhances the alpha IIb beta 3-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase.

    PubMed

    Zauli, G; Bassini, A; Vitale, M; Gibellini, D; Celeghini, C; Caramelli, E; Pierpaoli, S; Guidotti, L; Capitani, S

    1997-02-01

    The effect of thrombopoietin (TPO) on the functional activity of surface alpha IIb beta 3 (GPIIbIIIa) was investigated in both primary human megakaryocytic cells, derived from peripheral blood CD34+ cells, and HEL hematopoietic cell line. TPO (100 ng/mL) induced a sixfold to ninefold enhancement of adhesion of both primary megakaryocytic and HEL cells to plates coated with either fibrinogen or fibronectin and a parallel increase of immunoreactivity to the PAC1 monoclonal antibody (MoAb) and fluorescein isothiocyanate-fibrinogen, both of which recognize an activated state of alpha IIb beta 3. The enhanced adhesion to fibrinogen or fibronectin was mediated by the Arg-Gly-Asp (RGD) recognition sequence of alpha IIb beta 3, as it was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the alpha IIb beta subunit of alpha IIb beta 3 also inhibited cell attachment to fibrinogen or fibronectin, while MoAb to anti-alpha v beta 3 or anti-alpha 5 integrins were completely ineffective, clearly indicating that alpha IIb beta 3 participates in this association. A role for PI 3 kinase (PI 3-K) in the TPO-mediated increase in alpha IIb beta 3 function in megakaryocytic cells was suggested by the ability of the PI 3-K inhibitor wortmannin (100 nmol/L) and antisense oligonucleotides directed against the p85 regulatory subunit of PI 3-K to completely block the TPO-induced increase in alpha IIb beta 3 integrin activity upon TPO stimulation. The modulation of adhesiveness to extracellular matrix proteins containing the RGD motif mediated by TPO likely plays a physiologic role in megakaryocytopoiesis, as pretreatment of CD34+ cells with RGDS or anti-alpha IIb MoAb significantly reduced the number of megakaryocytic colonies obtained in a fibrinclot semisolid assay.

  18. FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

    PubMed

    Mak, Victor C Y; Wong, Oscar G W; Siu, Michelle K Y; Wong, Esther S Y; Ng, Wai-Yan; Wong, Richard W C; Chan, Ka-Kui; Ngan, Hextan Y S; Cheung, Annie N Y

    2015-07-01

    Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

  19. Phytosphingosine-1-phosphate stimulates chemotactic migration of L2071 mouse fibroblasts via pertussis toxin-sensitive G-proteins.

    PubMed

    Kim, Mi-Kyoung; Park, Kyoung Sun; Lee, Hyuck; Kim, Young Dae; Yun, Jeanho; Bae, Yoe-Sik

    2007-04-30

    Phytosphingosine-1-phosphate (PhS1P) was found to stimulate an intracellular calcium increase via phospholipase C but not pertussis toxin (PTX)-sensitive G-proteins in L2071 mouse fibroblasts. PhS1P also activated ERK and p38 kinase, and these activations by PhS1P were inhibited by PTX. Moreover, PhS1P stimulated the chemotactic migration of L2071 cells via PTX-sensitive Gi protein(s). In addition, the PhS1P-induced chemotactic migration of L2071 cells was also dramatically inhibited by LY294002 and SB203580 (inhibitors of phosphoinositide 3-kinase and p38 kinase, respectively). L2071 cells are known to express four S1P receptors, i.e., S1P1, S1P2, S1P3, and S1P4, and pretreatment with an S1P1 and S1P3 antagonist (VPC 23019) did not affect on PhS1P-induced chemotaxis. This study demonstrates that PhS1P stimulates at least two different signaling cascades, one is a PTX-insensitive but phospholipase C dependent intracellular calcium increase, and the other is a PTX-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and p38 kinase.

  20. Endomorphins delay constitutive apoptosis and alter the innate host defense functions of neutrophils.

    PubMed

    Azuma, Yasutaka; Ohura, Kiyoshi; Wang, Pao-Li; Shinohara, Mitsuko

    2002-04-01

    Recent studies have shown that opioid peptides are released from cells of the immune system during inflammation and stress, and are associated with altered immune responses. Moreover, concentrations of opioid peptides are increased in peripheral blood and at the sites of inflammatory reactions. The aim of this study was to evaluate immunological effects of opioid peptides endomorphins 1 and 2 on constitutive apoptosis, superoxide anion production, hydrogen peroxide production, adhesion, phagocytosis, and chemotaxis of neutrophils. Neutrophils were isolated by peritoneal lavage from rats. Endomorphins 1 and 2 significantly delayed constitutive neutrophil apoptosis. The delay of neutrophil apoptosis was markedly attenuated by LY294002, a phosphoinositide 3-kinase inhibitor. Moreover, endomorphins 1 and 2 activated the phosphoinositide 3-kinase pathway as determined by phosphorylation of BAD. In contrast, endomorphins 1 and 2 blocked the production of superoxide anion and hydrogen peroxide by PMA-stimulated neutrophils. In addition, endomorphins 1 and 2 inhibited neutrophil adhesion to fibronectin. Moreover, endomorphins 1 and 2 potentiated neutrophil chemotaxis toward zymosan-activated serum and IL-8, respectively. However, endomorphins 1 and 2 did not alter phagocytosis of Escherichia coli by neutrophils. These results suggest that endomorphins 1 and 2 may act to delay neutrophil apoptosis and alter the natural immune functions of neutrophils.

  1. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

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

    PubMed

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

    2016-03-01

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

  3. Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells.

    PubMed

    Jiménez, Eugenio; Pérez de la Blanca, Enrique; Urso, Loredana; González, Irene; Salas, Julián; Montiel, Mercedes

    2009-03-20

    Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.

  4. Breast Cancer Chemoresistance Mechanisms Through PI 3-Kinase and Akt Signaling

    DTIC Science & Technology

    2015-07-01

    PI3K/Akt pathway inhibitors including the PI3K inhibitor (BKM120) and the Akt inhibitor (MK2206) (Figure 13). Figure 12. Hyperactive (A) PI3K or...The PI3K/Akt pathway is hyperactive in more than 70% of breast tumors and is critical for tumor progression and resistance to anti-cancer drugs

  5. MiR-221 promotes IgE-mediated activation of mast cells degranulation by PI3K/Akt/PLCγ/Ca(2+) pathway.

    PubMed

    Xu, Hong; Gu, Li-Na; Yang, Qian-Yuan; Zhao, De-Yu; Liu, Feng

    2016-06-01

    Mast cells play a pivotal role in the immediate reaction in asthma. In a previous study, it was found that MicroRNA-221 (miR-221) was associated with asthma. Hence, in the present study, the role and the potential mechanisms of miR-221 on immunoglobulin E (IgE)-mediated activation of mast cells degranulation were investigated. MiR-221 expression was first quantified by qRT-PCR in IgE-mediated activation of mast cells. RBL-2H3 cells were then transfected with miR-221 mimic or miR-221 inhibitor, the IgE-mediated degranulation was detected in mast cells. The influence of miR-221 on expression of phospholipase C gamma (PLCγ1), p-PLCγ1, protein kinase B (Akt), phospho-Akt (p-Akt), inhibitor of kappa B (IκB-α), and phospho-IκB-α (p-IκB-α) were examined by Western blot, whereas free calcium ion (Ca(2+)) level was measured by flow cytometry and NF-κB expression was determined by EMSA. Phosphoinositide 3-kinase (PI3K)-inhibitor (LY294002) and NF-κB-inhibitor [pyrrolidine dithiocarbamate (PDTC)] were used to investigate the role of PI3K/Akt pathway and NF-κB in miR-221 promoting IgE-mediated activation of mast cells degranulation. The expression of miR-221 was upregulated in IgE-mediated activation of mast cells, and it was overexpressed in miR-221 mimic transfected cells. The degranulation was found to be significantly increased in miR-221 overexpressed cells while it was found to be significantly decreased in miR-221 downregulated cells. The expression of p-PLCγ1, p-Akt, p-IκB-α as well as NF-κB and Ca(2+) release were increased in miR-221 overexpressed cells. PI3K-inhibitor (LY294002) could rescue the promotion of degranulation caused by miR-221 in IgE-mediated activation of mast cells. However, NF-κB-inhibitor (PDTC) could not rescue the promotion of degranulation caused by miR-221 in IgE-mediated activation of mast cells. MiR-221 promotes IgE-mediated activation of mast cells degranulation by PI3K/Akt/PLCγ/Ca(2+) signaling pathway, in a non

  6. Co-Inhibition of GLUT-1 Expression and the PI3K/Akt Signaling Pathway to Enhance the Radiosensitivity of Laryngeal Carcinoma Xenografts In Vivo.

    PubMed

    Luo, Xing-Mei; Xu, Bin; Zhou, Min-Li; Bao, Yang-Yang; Zhou, Shui-Hong; Fan, Jun; Lu, Zhong-Jie

    2015-01-01

    In the present study, we investigated the role of GLUT-1 and PI3K/Akt signaling in radioresistance of laryngeal carcinoma xenografts. Volume, weight, radiosensitization, and the rate of inhibition of tumor growth in the xenografts were evaluated in different groups. Apoptosis was evaluated by TUNEL assay. In addition, mRNA and protein levels of GLUT-1, p-Akt, and PI3K in the xenografts were measured. Treatment with LY294002, wortmannin, wortmannin plus GLUT-1 AS-ODN, and LY294002 plus GLUT-1 AS-ODN after X-ray irradiation significantly reduced the size and weight of the tumors, rate of tumor growth, and apoptosis in tumors compared to that observed in the 10-Gy group (p<0.05). In addition, mRNA and protein expression of GLUT-1, p-Akt, and PI3K was downregulated. The E/O values of LY294002, LY294002 plus GLUT-1 AS-ODN, wortmannin, and wortmannin plus GLUT-1 AS-ODN were 2.7, 1.1, 1.8, and 1.8, respectively. Taken together, these data indicate that GLUT-1 AS-ODN as well as the inhibitors of PI3K/Akt signaling may act as radiosensitizers of laryngeal carcinoma in vivo.

  7. Targeting the Phosphatidylinositol-3-kinase Pathway in Gastric Cancer: Can Omics Improve Outcomes?

    PubMed Central

    Tran, Phu; Nguyen, Cham

    2016-01-01

    Phosphatidylinositol-3-kinase (PI3K) pathway signaling is an established oncogenic signal transduction pathway implicated in multiple malignancies. Therapeutic targeting of PI3K pathway components has improved outcomes in chronic lymphocytic leukemia, kidney cancer, breast cancer, and neuroendocrine tumors. Gastric cancers harbor some of the highest rates of oncogenic alterations in PI3K but attempts to translate this genomic observation have met with limited clinical success and novel approaches are needed. In the following review we discuss PI3K signaling, previous preclinical and clinical investigations in gastric cancer, and discuss future strategies aimed at overcoming resistance and improving efficacy. Identification and refinement of molecular tumor subtypes, development of predictive biomarkers along, and rational drug combination strategies are key to capitalizing on the therapeutic potential of PI3K pathway directed therapies in gastric cancers. PMID:27915478

  8. Measurement of phosphoinositide 3-kinase products in cultured Mammalian cells by HPLC.

    PubMed

    Cooke, Frank T

    2010-01-01

    The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explosion of interest in these PPIn. Although the four D-3 PPIn (phosphatidylinositol 3-phophate (PtdIns3P), PtdIns(3,4)P(2), PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3)) represent only a small proportion of PPIn, production of D-3 PPIn is required for an ever-increasing number of processes. Measurement of the PPIn levels in intact cells cultured cells has been vital to our understanding of the metabolism and function of these important signalling molecules; methods are described herein that allow measurement of PPIn levels in cultured cells, with emphasis on the 3-OH PPIn.

  9. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    SciTech Connect

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-16

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  10. Sesamin induces melanogenesis by microphthalmia-associated transcription factor and tyrosinase up-regulation via cAMP signaling pathway.

    PubMed

    Jiang, Zequn; Li, Shasha; Liu, Yunyi; Deng, Pengyi; Huang, Jianguo; He, Guangyuan

    2011-10-01

    In this study, we confirmed that sesamin, an active lignan isolated from sesame seed and oil, is a novel skin-tanning compound. The melanin content and tyrosinase activity were increased by sesamin in a dose-dependent manner in B16 melanoma cells. The mRNA and protein levels of tyrosinase were also enhanced after the treatment with sesamin. Western blot analysis revealed that sesamin induced and sustained up-regulation of microphthalmia-associated transcription factor (MITF). Sesamin could activate cAMP response element (CRE) binding protein (CREB), but it had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or Akt. Moreover, sesamin activated protein kinase A (PKA) via a cAMP-dependent pathway. Consistent with these results, sesamin-mediated increase of melanin synthesis was reduced significantly by H-89, a PKA inhibitor, but not by SB203580, a p38 MAPK inhibitor or by LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Sesamin-mediated phosphorylation of CREB and induction of MITF and tyrosinase expression were also inhibited by H-89. These findings indicated that sesamin could stimulate melanogenesis in B16 cells via the up-regulation of MITF and tyrosinase, which was, in turn, due to the activation of cAMP signaling.

  11. Subtilisin-like proprotein convertase PACE4 is required for skeletal muscle differentiation.

    PubMed

    Yuasa, Keizo; Masuda, Tetsuya; Yoshikawa, Chihiro; Nagahama, Masami; Matsuda, Yoshiko; Tsuji, Akihiko

    2009-09-01

    Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expression of shRNA targeting PACE4, but not furin, suppressed the expression of the muscle-specific myosin light chain (MLC). Interestingly, reduced expression of MLC was restored following treatment with recombinant mature IGF-II. Finally, we demonstrated that the PI3K inhibitor LY294002 blocked the induction of PACE4 mRNA, a result not observed when another myogenic differentiation inhibitor, SB203580 (p38 MAP kinase inhibitor), was employed, indicating the presence of a positive feedback loop regulating PACE4 expression. These results suggest that PACE4 plays an important role in myogenic differentiation through its association with the IGF-II pathway.

  12. Interleukin-21 induces migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

    PubMed

    Xing, R; Jin, Y; Sun, L; Yang, L; Li, C; Li, Z; Liu, X; Zhao, J

    2016-05-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast-like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)-21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL-21 on the migration, invasion and matrix metalloproteinase (MMP) expression in RA-FLS. We found that IL-21 promoted the migration, invasion and MMP (MMP-2, MMP-3, MMP-9, MMP-13) production in RA-FLS. Moreover, IL-21 induced activation of the phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription-3 (STAT-3) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, and blockage of these pathways [PI3K/protein kinase B (AKT) inhibitor LY294002, STAT-3 inhibitor STA-21 and ERK1/2 inhibitor PD98059] attenuated IL-21-induced migration and secretion of MMP-3 and MMP-9. In conclusion, our results suggest that IL-21 promotes migration and invasion of RA-FLS. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA.

  13. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures.

    PubMed

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-08-01

    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders.

  14. The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus

    SciTech Connect

    Pendergrass, Karl D.; Gwathmey, TanYa M.; Michalek, Ryan D.; Grayson, Jason M.; Chappell, Mark C.

    2009-06-26

    We and others have reported significant expression of the Ang II Type 1 receptor (AT1R) on renal nuclei; thus, the present study assessed the functional pathways and distribution of the intracellular AT1R on isolated nuclei. Ang II (1 nM) stimulated DCF fluorescence, an intranuclear indicator of reactive oxygen species (ROS), while the AT1R antagonist losartan or the NADPH oxidase (NOX) inhibitor DPI abolished the increase in ROS. Dual labeling of nuclei with antibodies against nucleoporin 62 (Nup62) and AT1R or the NADPH oxidase isoform NOX4 revealed complete overlap of the Nup62 and AT1R (99%) by flow cytometry, while NOX4 was present on 65% of nuclei. Treatment of nuclei with a PKC agonist increased ROS while the PKC inhibitor GF109203X or PI3 kinase inhibitor LY294002 abolished Ang II stimulation of ROS. We conclude that the Ang II-AT1R-PKC axis may directly influence nuclear function within the kidney through a redox sensitive pathway.

  15. Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication.

    PubMed

    Huang, Xiaohong; Wang, Wei; Huang, Youhua; Xu, Liwen; Qin, Qiwei

    2014-12-01

    Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

  16. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. Part 2: PI3K/Akt/PTEN, mTOR, SHH/PTCH and angiogenesis.

    PubMed

    Newton, Herbert B

    2004-02-01

    Brain tumors are a diverse group of malignancies that remain refractory to conventional treatment approaches. Molecular neuro-oncology has now begun to clarify the transformed phenotype of brain tumors and identify oncogenic pathways that might be amenable to targeted therapy. Activity of the phosphoinositide 3; kinase (PI3K)/Akt pathway is often upregulated in brain tumors due to excessive stimulation by growth factor receptors and Ras. Loss of function of the tumor suppressor gene PTEN also frequently contributes to upregulation of PI3K/Akt. Several compounds, such as wortmannin and LY-294002, can target PI3K and inhibit activity of this pathway. The mammalian target of rapamycin (mTOR) is an important regulator of cell growth and metabolism and is often upregulated by Akt. Clinical trials of CCI-779, an inhibitor of mTOR, are ongoing in recurrent malignant glioma patients. The sonic hedgehog/PTCH pathway is involved in the tumorigenesis of some familial and sporadic medulloblastomas. This pathway can be targeted by cyclopamine, which is under evaluation in preclinical studies. Angiogenesis is a critical process for development and progression of brain tumors. Targeted approaches to inhibit angiogenesis include monoclonal antibodies, receptor tyrosine kinase inhibitors, antisense oligonucleotides and gene therapy. Clinical trials are ongoing for numerous angiogenesis inhibitors, including thalidomide, CC-5103 and PTK 787/ZK 222584. Further development of targeted therapies and evaluation of these new agents in clinical trials will be needed to improve survival and quality of life of patients with brain tumors.

  17. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways

    PubMed Central

    Li, Mingwei; Sun, Xuefei; Ma, Liang; Jin, Lu; Zhang, Wenfei; Xiao, Min; Yu, Qing

    2017-01-01

    SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis. PMID:28067275

  18. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways.

    PubMed

    Li, Mingwei; Sun, Xuefei; Ma, Liang; Jin, Lu; Zhang, Wenfei; Xiao, Min; Yu, Qing

    2017-01-09

    SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis.

  19. Over-Expression of PDGFR-β Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

    PubMed Central

    Li, Wei; Zhao, Xiaohui; Yu, Yang; Zhu, Jinkun; Qin, Zhexue; Wang, Qiang; Wang, Kui; Lu, Wei; Liu, Jie; Huang, Lan

    2012-01-01

    The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes. PMID:22355314

  20. LINGO-1 receptor promotes neuronal apoptosis by inhibiting WNK3 kinase activity.

    PubMed

    Zhang, Zhaohuan; Xu, Xiaohui; Xiang, Zhenghua; Yu, Zhongwang; Feng, Jifeng; He, Cheng

    2013-04-26

    LINGO-1 is a functional component of the Nogo receptor 1 · p75(NTR) · LINGO-1 and Nogo receptor 1 · TAJ (TNFRSF19/TROY)·LINGO-1 signaling complexes. It has recently been shown that LINGO-1 antagonists significantly improve neuronal survival after neural injury. However, the mechanism by which LINGO-1 signaling influences susceptibility to apoptosis remains unknown. In an effort to better understand how LINGO-1 regulates these signaling pathways, we used an established model of serum deprivation (SD) to induce neuronal apoptosis. We demonstrate that treatment either with a construct containing the intracellular domain of LINGO-1 or with Nogo66, a LINGO-1 receptor complex agonist, resulted in an enhanced rate of apoptosis in primary cultured cortical neurons under SD. Reducing the expression levels of the serine/threonine kinase WNK3 using shRNA or inhibiting its kinase activity had similar effects on the survival of serum-deprived neurons. Consistent with these observations, we found that LINGO-1 and WNK3 co-localized and co-precipitated in cultured cortical neurons and brain tissue. Significantly, this co-association was enhanced by Nogo66 treatment. Binding of WNK3 to the intracellular domain of LINGO-1 led to a reduction in WNK3 kinase activity, as did Nogo66 stimulation. Moreover, in vitro and in vivo evidence indicates that endogenous WNK3 suppresses SD-induced neuronal apoptosis in a kinase-dependent manner, as the expression of either a WNK3 RNAi construct or a kinase-dead N-terminal fragment of WNK3 led to increased apoptosis. Taken together, our results show that LINGO-1 potentiates neuronal apoptosis, likely by inhibiting WNK3 kinase activity.

  1. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  2. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  3. Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells.

    PubMed

    Del Viscovo, Adelaide; Secondo, Agnese; Esposito, Alba; Goglia, Fernando; Moreno, Maria; Canzoniero, Lorella M T

    2012-06-01

    The role of 3,5,3'-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca(2+) concentration ([Ca(2+)](i)) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH(3) cells in the absence or presence of extracellular Ca(2+). When applied in Ca(2+)-free solution, T2 and T3 increased [Ca(2+)](i), in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N(G)-nitro-l-arginine methyl ester and l-n(5)-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca(2+)](i) increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca(2+) stores did not interfere with the T2- and T3-induced [Ca(2+)](i) increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca(2+)](i) and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca(2+)](i) increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na(+)/Ca(2+) exchanger by CGP37157 significantly reduced the [Ca(2+)](i) increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca(2+)](i) and intracellular Na(+) concentration; nimodipine reduced the [Ca(2+)](i) increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na(+)/H(+) pump by 5-(N-ethyl-N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na(+)/Ca(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short

  4. Hepatoprotective Effect of Quercetin on Endoplasmic Reticulum Stress and Inflammation after Intense Exercise in Mice through Phosphoinositide 3-Kinase and Nuclear Factor-Kappa B

    PubMed Central

    Tang, Yuhan; Li, Juan; Gao, Chao; Xu, Yanyan; Li, Yanyan; Yu, Xiao; Wang, Jing; Liu, Liegang

    2016-01-01

    The mechanisms underlying intense exercise-induced liver damage and its potential treatments remain unclear. We explored the hepatoprotection and mechanisms of quercetin, a naturally occurring flavonoid, in strenuous exercise-derived endoplasmic reticulum stress (ERS) and inflammation. Intense exercise (28 m/min at a 5° slope for 90 min) resulted in the leakage of aminotransferases in the BALB/C mice. The hepatic ultrastructural malformations and oxidative stress levels were attenuated by quercetin (100 mg/kg·bw). Intense exercise and thapsigargin- (Tg-) induced ERS (glucose-regulated protein 78, GRP78) and inflammatory cytokines levels (IL-6 and TNF-α) were decreased with quercetin. Furthermore, quercetin resulted in phosphoinositide 3-kinase (PI3K) induction, Ca2+ restoration, and blockade of the activities of Jun N-terminal kinase (JNK), activating transcription factor 6 (ATF6) and especially NF-κB (p65 and p50 nuclear translocation). A PI3K inhibitor abrogated the protection of quercetin on ERS and inflammation of mouse hepatocytes. SP600125 (JNK inhibitor), AEBSF (ATF6 inhibitor), and especially PDTC (NF-κB inhibitor) enhanced the quercetin-induced protection against Tg stimulation. Collectively, intense exercise-induced ERS and inflammation were attenuated by quercetin. PI3K/Akt activation and JNK, ATF6, and especially NF-κB suppression were involved in the protection. Our results highlight a novel preventive strategy for treating ERS and inflammation-mediated liver damage induced by intense exercise using natural phytochemicals. PMID:27504150

  5. Dual Inhibiton of Bruton's Tyrosine Kinase and Phosphoinositide-3-Kinase p110δ as a Therapeutic Approach to Treat non-Hodgkin's B cell Malignancies.

    PubMed

    Alfaro, Jennifer; Perez de Arce, Felipe; Belmar, Sebastian; Fuentealba, Glenda; Avila, Patricio; Ureta, Gonzalo; Flores, Camila; Acuna, Claudia; Delgado, Luz; Gaete, Diana; Pujala, Brahmam; Barde, Anup; Nayak, Anjan K; Upendra, Tvr; Patel, Dhananjay; Chauhan, Shailender; Sharma, Vijay K; Kanno, Stacy; Almirez, Ramona G; Hung, David T; Chakravarty, Sarvajit; Rai, Roopa; Bernales, Sebastian; Quinn, Kevin P; Pham, Son M; McCullagh, Emma

    2017-03-15

    Although new targeted therapies such as ibrutinib and idelalisib have made a large impact on non-Hodgkin's lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors and possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We have discovered a single molecule MDVN1003 that inhibits Bruton's tyrosine kinase (BTK) and phosphatidylinositol-3-kinase delta (PI3Kδ), two proteins regulated by the B cell receptor (BCR) that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK 1/2), two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

  6. Class II phosphoinositide 3-kinase C2β regulates a novel signaling pathway involved in breast cancer progression

    PubMed Central

    Abbott, Jonathan J.; Piñeiro, Roberto; Buus, Richard; Iezzi, Manuela; Ricci, Francesca; Bergamaschi, Daniele; Ostano, Paola; Chiorino, Giovanna; Lattanzio, Rossano; Broggini, Massimo; Piantelli, Mauro; Maffucci, Tania; Falasca, Marco

    2016-01-01

    It is now well established that the enzymes phosphoinositide 3-kinases (PI3Ks) have a key role in the development and progression of many cancer types and indeed PI3Ks inhibitors are currently being tested in clinical trials. Although eight distinct PI3K isoforms exist, grouped into three classes, most of the evidence currently available are focused on one specific isoform with very little known about the potential role of the other members of this family in cancer. Here we demonstrate that the class II enzyme PI3K-C2β is overexpressed in several human breast cancer cell lines and in human breast cancer specimens. Our data indicate that PI3K-C2β regulates breast cancer cell growth in vitro and in vivo and that PI3K-C2β expression in breast tissues is correlated with the proliferative status of the tumor. Specifically we show that downregulation of PI3K-C2β in breast cancer cell lines reduces colony formation, induces cell cycle arrest and inhibits tumor growth, in particular in an estrogen-dependent in vivo xenograft. Investigation of the mechanism of the PI3K-C2β-dependent regulation of cell cycle progression and cell growth revealed that PI3K-C2β regulates cyclin B1 protein levels through modulation of microRNA miR-449a levels. Our data further demonstrate that downregulation of PI3K-C2β inhibits breast cancer cell invasion in vitro and breast cancer metastasis in vivo. Consistent with this, PI3K-C2β is highly expressed in lymph-nodes metastases compared to matching primary tumors. These data demonstrate that PI3K-C2β plays a pivotal role in breast cancer progression and in metastasis development. Our data indicate that PI3K-C2β may represent a key molecular switch that regulates a rate-limiting step in breast tumor progression and therefore it may be targeted to limit breast cancer spread. PMID:26934321

  7. Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

    PubMed Central

    Marty, Bérengère; Maire, Virginie; Gravier, Eléonore; Rigaill, Guillem; Vincent-Salomon, Anne; Kappler, Marion; Lebigot, Ingrid; Djelti, Fathia; Tourdès, Audrey; Gestraud, Pierre; Hupé, Philippe; Barillot, Emmanuel; Cruzalegui, Francisco; Tucker, Gordon C; Stern, Marc-Henri; Thiery, Jean-Paul; Hickman, John A; Dubois, Thierry

    2008-01-01

    Introduction Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs. Methods In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines. Results The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition. Conclusions These data provide insight into the molecular pathogenesis of BLCs and implicate the

  8. Sphingosine kinase: a point of convergence in the action of diverse neutrophil priming agents.

    PubMed

    MacKinnon, Alison C; Buckley, Avril; Chilvers, Edwin R; Rossi, Adriano G; Haslett, Christopher; Sethi, Tariq

    2002-12-01

    Neutrophils are a vital component of the early acute inflammatory response, but can cause profound tissue damage when activated to excess or prevented from undergoing apoptosis. However, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. The structurally diverse neutrophil-priming agents platelet-activating factor, TNF-alpha, and the substance P analog [D-Arg(6), D-Trp(7,9),N(me)Phe(8)]-substance P(6-11) (SP-G) stimulated a rapid increase in sphingosine kinase activity in freshly isolated human neutrophils. This activity was blocked by preincubation with the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS). DMS also inhibited the increase in intracellular calcium concentration stimulated by platelet-activating factor, fMLP, and SP-G. This suggests that the increase in intracellular calcium concentration by these agents is dependent on sphingosine kinase activation and the generation of sphingosine-1-phosphate. Changes in cell polarization and the augmentation of the fMLP-induced superoxide anion generation, by all priming agents were also inhibited by DMS, while only the superoxide anion release was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Moreover, SP-G and GM-CSF inhibited constitutive neutrophil apoptosis which was completely blocked by DMS. These results suggest a novel role for sphingosine kinase in the regulation of neutrophil priming.

  9. 2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulates proliferation of HAPI microglia by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.

    PubMed

    Xu, Guangfei; Li, Yuanye; Yoshimoto, Katsuhiko; Wu, Qiyun; Chen, Gang; Iwata, Takeo; Mizusawa, Noriko; Wan, Chunhua; Nie, Xiaoke

    2014-01-30

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that induces apoptosis of neurons and a pro-inflammatory response in microglial cells. First, we found that TCDD induced proliferation of HAPI microglial cells in a dose- and time-dependent manner. Flow cytometry analysis showed that this proliferation by TCDD was due to mainly enhancing the G1 to S phase transition. Next, it was found that TCDD treatment led to up-regulation of cyclin D1, which induces cell cycle progression from G1 to S phase, in a time-dependent manner. As for molecular mechanism, we revealed that TCDD was capable of inducing Akt phosphorylation and activation, resulting in phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β). Inactivated GSK-3β attenuated proteasomal degradation of cyclin D1 by reducing Thr(286)-phosphorylated cyclin D1 levels. Moreover, inactivated GSK-3β increased cyclin D1 gene transcription by increasing its transcription factor β-catenin in the nucleus. Further, blockage of phosphoinositide 3-kinase/Akt kinase with their specific inhibitors, LY294002 and Akt 1/2 kinase inhibitor, significantly reduced TCDD-enhanced proliferation of HAPI microglial cells. In conclusion, TCDD stimulates proliferation of HAPI microglial cells by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.

  10. Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting.

    PubMed

    Fiory, Francesca; Oriente, Francesco; Miele, Claudia; Romano, Chiara; Trencia, Alessandra; Alberobello, Anna Teresa; Esposito, Iolanda; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2004-03-19

    We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.

  11. Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPARβ/δ in HepG2 cells.

    PubMed

    Luo, Guanghua; Shi, Yuanping; Zhang, Jun; Mu, Qinfeng; Qin, Li; Zheng, Lu; Feng, Yuehua; Berggren-Söderlund, Maria; Nilsson-Ehle, Peter; Zhang, Xiaoying; Xu, Ning

    2014-02-28

    It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPARβ/δ pathway.

  12. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids

    PubMed Central

    Limbourg, Florian P.; Huang, Zhihong; Plumier, Jean-Christophe; Simoncini, Tommaso; Fujioka, Masayuki; Tuckermann, Jan; Schütz, Günther; Moskowitz, Michael A.; Liao, James K.

    2002-01-01

    Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS–/– mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF. PMID:12464678

  13. PDGF-induced PI3K-mediated signaling enhances the TGF-β-induced osteogenic differentiation of human mesenchymal stem cells in a TGF-β-activated MEK-dependent manner.

    PubMed

    Yokota, Jun; Chosa, Naoyuki; Sawada, Shunsuke; Okubo, Naoto; Takahashi, Noriko; Hasegawa, Tomokazu; Kondo, Hisatomo; Ishisaki, Akira

    2014-03-01

    Transforming growth factor-β (TGF-β) is a critical regulator of osteogenic differentiation and the platelet-derived growth factor (PDGF) is a chemoattractant or mitogen of osteogenic mesenchymal cells. However, the combined effects of these regulators on the osteogenic differentiation of mesenchymal cells remains unknown. In this study, we investigated the effects of TGF-β and/or PDGF on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The TGF-β-induced osteogenic differentiation of UE7T-13 cells, a bone marrow-derived hMSC line, was markedly enhanced by PDGF, although PDGF alone did not induce differentiation. TGF-β induced extracellular signal-regulated kinase (ERK) phosphorylation and PDGF induced Akt phosphorylation. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, suppressed the osteogenic differentiation induced by TGF-β alone. Moreover, U0126 completely suppressed the osteogenic differentiation synergistically induced by TGF-β and PDGF, whereas the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002, only partially suppressed this effect. These results suggest that the enhancement of TGF-β-induced osteogenic differentiation by PDGF-induced PI3K/Akt-mediated signaling depends on TGF-β-induced MEK activity. Thus, PDGF positively modulates the TGF-β-induced osteogenic differentiation of hMSCs through synergistic crosstalk between MEK- and PI3K/Akt-mediated signaling.

  14. Alzheimer's disease-type neuronal tau hyperphosphorylation induced by Aβ oligomers

    PubMed Central

    De Felice, Fernanda G.; Wu, Diana; Lambert, Mary P.; Fernandez, Sara J.; Velasco, Pauline T.; Lacor, Pascale N.; Bigio, Eileen H.; Jerecic, Jasna; Acton, Paul J.; Shughrue, Paul J.; Chen-Dodson, Elizabeth; Kinney, Gene G.; Klein, William L.

    2008-01-01

    Alzheimer’s disease (AD) is characterized by presence of extracellular fibrillar Aβ in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble Aβ oligomers (ADDLs). A major question is how these disparate facets of AD pathology are mechanistically related. Here we show that, independent of the presence of fibrils, ADDLs stimulate tau phosphorylation in mature cultures of hippocampal neurons and in neuroblastoma cells at epitopes characteristically hyperphosphorylated in AD. A monoclonal antibody that targets ADDLs blocked their attachment to synaptic binding sites and prevented tau hyperphosphorylation. Tau phosphorylation was blocked by the Src family tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide (PP1), and by the phosphatidylinositol-3-kinase inhibitor LY294002. Significantly, tau hyperphosphorylation was also induced by a soluble aqueous extract containing Aβ oligomers from AD brains, but not by an extract from non-AD brains. Aβ oligomers have been increasingly implicated as the main neurotoxins in AD, and the current results provide a unifying mechanism in which oligomer activity is directly linked to tau hyperphosphorylation in AD pathology. PMID:17403556

  15. Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish.

    PubMed

    Hirohashi, Noritaka; Harada, Kaori; Chiba, Kazuyoshi

    2008-06-01

    Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.

  16. Genetic depletion of glutathione peroxidase-1 potentiates nephrotoxicity induced by multiple doses of cocaine via activation of angiotensin II AT1 receptor.

    PubMed

    Mai, Huynh Nhu; Chung, Yoon Hee; Shin, Eun-Joo; Kim, Dae-Joong; Jeong, Ji Hoon; Nguyen, Thuy-Ty Lan; Nam, Yunsung; Lee, Yu Jeung; Nah, Seung-Yeol; Yu, Dae-Yeul; Jang, Choon-Gon; Ho, Ye-Shih; Lei, Xin Gen; Kim, Hyoung-Chun

    2016-01-01

    We investigated the possible roles of angiotensin II type 1 receptor (AT1R) and oxidative stress responsive nuclear factor κB (NFκB) in renal damage caused by multiple doses of cocaine in glutathione peroxidase (GPx)-1 gene-depleted mice. Treatment with cocaine resulted in significant increases in malondialdehyde, protein carbonyl, and pro-apoptotic Bax expression and decreases in the ratio of glutathione (GSH) and its oxidized form (GSSG), GSH-dependent enzymes, and anti-apoptotic factors in the kidney. These alterations were more pronounced in GPx-1 knockout (-/-) mice than in wild type (WT) mice. Notably, the AT1R antagonist losartan protected against the renal toxicity induced by cocaine, whereas the NFκB inhibitor pyrrolidine dithiocarbamate was not protective. The toxicity was more pronounced in GPx-1 (-/-) mice than in WT mice. The protective effect afforded by losartan against cocaine toxicity appeared to be more sensitive in GPx-1 (-/-) mice than that in WT mice. These losartan-mediated protective effects were inhibited by the phosphatidyl-inositol-3-kinase (PI3K) inhibitor LY294002, indicating that losartan provides significant protection from cocaine-induced renal toxicity through PI3K/Akt signaling. Our results suggest that genetic inhibition of GPx-1 potentiates cocaine-induced renal damage via activation of AT1R by inhibition of PI3K-Akt signaling, and that AT1R can be a therapeutic target against renal toxicity induced by cocaine.

  17. Geniposide induces the expression of heme oxygenase-1 via PI3K/Nrf2-signaling to enhance the antioxidant capacity in primary hippocampal neurons.

    PubMed

    Yin, Fei; Liu, Jianhui; Zheng, Xuxu; Guo, Lixia; Xiao, He

    2010-01-01

    Oxidative stress in brain is emerging as a potential causal factor in aging and age-related neurodegenerative disorders. A large body of evidence shows that induction of endogenous antioxidative proteins seems to be a reasonable strategy for delaying the progression of cell injury. In this study, geniposide upregulates the expression of heme oxygenase-1 (HO-1) to attenuate the cell apoptosis induced by 3-morpholinosydnonimine hydrochloride (SIN-1) in primary cultured hippocampal neurons. Furthermore, geniposide induces the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and activation of phosphatidylinositol 3'-kinase (PI3K) in the presence of oxidative stress, and both LY294002 (a specific inhibitor of PI3K) and Zinc protoporphyrin (ZnPP, an inhibitor of HO-1) decrease the cytoprotective action of geniposide in hippocampal neurons. Taken together, the novel cytoprotective mechanism of geniposide to antagonize oxidative stress may be involved in PI3K- and Nrf2-mediated upregulation of the antioxidative enzyme HO-1.

  18. Low Doses of Camptothecin Induced Hormetic and Neuroprotective Effects in PC12 Cells

    PubMed Central

    Zhang, Chao; Chen, Shenghui; Bao, Jiaolin; Zhang, Yulin; Huang, Borong; Jia, Xuejing; Chen, Meiwan; Wan, Jian-Bo; Su, Huanxing; Wang, Yitao

    2015-01-01

    Hormetic response is an adaptive mechanism for a cell or organism surviving in an unfavorable environment. It has been an intriguing subject of researches covering a broad range of biological and medical disciplines, in which the underlying significance and molecular mechanisms are under intensive investigation. In the present study, we demonstrated that topoisomerase I inhibitor camptothecin (CPT), a potent anticancer agent, induced an obvious hormetic response in rat pheochromocytoma PC12 cells. Camptothecin inhibited PC12 cell growth at relative high doses as generally acknowledged while stimulated the cell growth by as much as 39% at low doses. Moreover, low doses of CPT protected the cells from hydrogen peroxide (H2O2)-induced cell death. Phosphoinositide 3-kinase (PI3K)/Akt and nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathways were reported playing pivotal roles in protecting cells from oxidative stress. We observed that these 2 pathways were upregulated by low doses of CPT, as evidenced by increased levels of phosphorylated PI3K, phosphorylated Akt, phosphorylated mammalian target of rapamycin, Nrf2, and HO-1; and abolishment of the growth-promoting and neuroprotective effects of CPT by LY294002, a PI3K inhibitor. These results suggest that the hormetic and neuroprotective effects of CPT at low doses on PC12 cells were attributable, at least partially, to upregulated PI3K/Akt and Nrf2/HO-1 pathways. PMID:26674066

  19. The effects of adiponectin and leptin on human endothelial cell proliferation: a live-cell study.

    PubMed

    Alvarez, Granada; Visitación Bartolomé, M; Miana, María; Jurado-López, Raquel; Martín, Ruben; Zuluaga, Pilar; Martinez-Martinez, Ernesto; Nieto, M Luisa; Alvarez-Sala, Luis A; Millán, Jesús; Lahera, Vicente; Cachofeiro, Victoria

    2012-01-01

    The effect of adiponectin and leptin on the proliferation of the human microvascular endothelial cell line (HMEC-1) was studied in the absence or presence of fetal bovine serum (FBS). The participation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) pathways in this effect were evaluated. We studied the effect of both adipokines on the motility, mitosis, proliferation and cell death processes of HMEC-1 cells using live-cell imaging techniques. Adiponectin but not leptin further increased the proliferative effect induced by FBS on HMEC-1. This effect seems to be the consequence of an increase in the mitotic index in adiponectin-treated cells when compared to untreated ones. The presence of either the mitogen-activated protein kinase (MAPK) inhibitor (PD98059), or PI-3K inhibitor (LY294002), reduced the effect of adiponectin in a dose-dependent manner. Neither adipokine was able to affect HMEC-1 proliferation in FBS-free conditions. Duration of mitosis, cell motility and the cell death process were similar in all conditions. These data suggest that adiponectin and leptin exert different effects on endothelial cell function. Adiponectin was able to potentiate proliferation of HMEC-1. This effect involves the activation of both PI3-K/Akt and ERK/MAPK pathways. However, it seems to exert minimal effects on HMEC-1 function in the case of leptin.

  20. Neuregulin-1 attenuates doxorubicin-induced autophagy in neonatal rat cardiomyocytes.

    PubMed

    An, Tao; Huang, Yan; Zhou, Qiong; Wei, Bing Qi; Zhang, Rong Cheng; Yin, Shi Jie; Zou, Chang Hong; Zhang, Yu Hui; Zhang, Jian

    2013-08-01

    Recombinant human neuregulin-1 (rhNRG-1) improves cardiac function in animal models of doxorubicin (DOX)-induced cardiomyopathy, but the underlying mechanism remains largely unknown. Here, we confirm a role for rhNRG-1 in attenuating DOX-induced autophagy and define the signaling pathways through which it mediates some of its effects. Neonatal rat ventricular myocytes were subjected to different treatments both to induce autophagy and to determine the effects of rhNRG-1 on the process. The rhNRG-1 inhibited DOX-induced autophagy, reduced reactive oxygen species production and increased protein expression of Bcl-2, effects that were recapitulated when the cells were treated with the antioxidant N-acetylcysteine. These effects were blocked by the phosphatidylinositol 3-kinase inhibitor LY294002, pointing to the involvement of the Akt pathway in mediating the process. Inhibition of Bcl-2 expression with small interfering RNA silencing also inhibited rhNRG-1's ability to attenuate DOX-induced autophagy. The rhNRG-1 is a potent inhibitor of DOX-induced autophagy and multiple signaling pathways, including Akt and activation of reactive oxygen species, play important roles in the anti-autophagy effect. The rhNRG-1 is a novel drug that may be effectively therapeutically in protecting further damage in DOX-induced damaged myocardium.

  1. Activation of the MAP Kinase Cascade by Exogenous Calcium-Sensing Receptor

    SciTech Connect

    Hobson, Susan A.; Wright, Jay W.; Lee, Fred; Mcneil, Scott; Bilderback, Tim R.; Rodland, Karin D.

    2003-02-01

    In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled Calcium-sensing Receptor (CaR), as expression of the non-functional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3' kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR-mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses.

  2. Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.

    PubMed

    Salaün, Patrick; Le Breton, Magali; Morales, Julia; Bellé, Robert; Boulben, Sandrine; Mulner-Lorillon, Odile; Cormier, Patrick

    2004-06-10

    In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.

  3. GnRH regulates trophoblast invasion via RUNX2-mediated MMP2/9 expression

    PubMed Central

    Peng, Bo; Zhu, Hua; Klausen, Christian; Ma, Liyang; Wang, Yan-ling; Leung, Peter C.K.

    2016-01-01

    STUDY HYPOTHESIS We hypothesized that Runt-related transcription factor 2 (RUNX2), matrix metalloproteinase (MMP)2 and MMP9 are involved in basal and gonadotrophin-releasing hormone (GnRH)-induced human extravillous trophoblast (EVT) cell invasion. STUDY FINDING Our finding indicates that GnRH-induced RUNX2 expression enhances the invasive capacity of EVT cells by modulating the expression of MMP2 and MMP9. WHAT IS KNOWN ALREADY GnRH is expressed in first-trimester placenta and exerts pro-invasive effects on EVT cells in vitro. RUNX2 regulates MMP2 and MMP9 expression and is often associated with invasive phenotypes. STUDY DESIGN, SAMPLES/MATERIALS, METHODS First-trimester human placenta (n = 9) was obtained from women undergoing elective termination of pregnancy. The localization of RUNX2, MMP2 and MMP9 in first-trimester human placenta was examined by immunohistochemistry. Primary or immortalized (HTR-8/SVneo) EVT cells were treated alone or in combination with GnRH, GnRH antagonist Antide, MAPK kinase inhibitor PD98095, phosphatidylinositol 3-kinase inhibitor LY294002, MMP2/9 inhibitor or small interfering RNAs (siRNAs) targeting RUNX2, MMP2 and/or MMP9. Protein and mRNA levels were measured by western blot and RT–PCR, respectively. Cell invasiveness was evaluated by transwell Matrigel or collagen I invasion assays. MAIN RESULTS AND THE ROLE OF CHANCE RUNX2, MMP2 and MMP9 were detected in the cell column regions of human first-trimester placental villi. GnRH treatment increased RUNX2 mRNA and protein levels in HTR-8/SVneo cells and primary EVTs, and these effects were attenuated by co-treatment with Antide, PD98095 or LY294002. Down-regulation of RUNX2 by siRNA reduced basal and GnRH-induced MMP2/9 expression and cell invasion. Moreover, pharmacological inhibition or siRNA-mediated knockdown of MMP2/9 reduced basal and GnRH-induced cell invasion. LIMITATIONS, REASONS FOR CAUTION The lack of an in vivo model is the major limitation of our in vitro study. WIDER

  4. Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.

    PubMed Central

    Folli, F; Saad, M J; Backer, J M; Kahn, C R

    1993-01-01

    Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse. Images PMID:7691886

  5. Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase.

    PubMed

    Nikolic, Dejan M; Cholewa, Jill; Gass, Cecelia; Gong, Ming C; Post, Steven R

    2007-04-01

    Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A-/- macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting G(i/o) activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.

  6. Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product.

    PubMed

    Kim, H H; Sierke, S L; Koland, J G

    1994-10-07

    The ErbB3 protein is a member of the ErbB subfamily of receptor protein tyrosine kinases. In the present study, the mechanism by which the ErbB3 protein is phosphorylated and the signal-transducing functions of this phosphorylated protein were investigated. When phosphorylated by the epidermal growth factor receptor in vitro, the ErbB3 protein strongly associated with the regulatory p85 subunit and the catalytic activity of phosphatidylinositol (PI) 3-kinase. The association of PI 3-kinase with ErbB3 in human breast cancer cells was found to be correlated with the constitutive phosphorylation of ErbB3 on tyrosine residues. In MDA-MB-468 breast cancer cells in which the ErbB3 protein is not constitutively phosphorylated, stimulation with epidermal growth factor led to the phosphorylation of ErbB3 on tyrosine residues and the formation of a functional signal transduction complex involving the ErbB3 protein and PI 3-kinase. These results suggest that the ErbB3 protein can be phosphorylated on tyrosine residues by a cross-phosphorylation mechanism and that the phosphorylated ErbB3 protein can couple other growth factor receptor protein tyrosine kinases to the PI 3-kinase pathway in a manner similar to the insulin receptor substrate 1 protein.

  7. Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt signaling in fibroblasts.

    PubMed

    Park, Chang Shin; Schneider, Ian C; Haugh, Jason M

    2003-09-26

    Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3'-phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110alpha catalytic subunit of PI 3-kinase contributes most to PDGF-stimulated 3'-PI production. Thus, at high concentrations of PDGF the kinetics of 3'-PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.

  8. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  9. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling

    PubMed Central

    Cinnamon, Einat; Sawala, Annick; Tittiger, Claus; Paroush, Ze'ev

    2016-01-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  10. Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

    PubMed

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-21

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

  11. Vav3 Modulates B Cell Receptor Responses by Regulating Phosphoinositide 3-Kinase Activation

    PubMed Central

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M.; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-01

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux. PMID:11805146

  12. Class IA phosphoinositide 3-kinase regulates heart size and physiological cardiac hypertrophy.

    PubMed

    Luo, Ji; McMullen, Julie R; Sobkiw, Cassandra L; Zhang, Li; Dorfman, Adam L; Sherwood, Megan C; Logsdon, M Nicole; Horner, James W; DePinho, Ronald A; Izumo, Seigo; Cantley, Lewis C

    2005-11-01

    Class I(A) phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110alpha catalytic subunit of class I(A) PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand the role of class I(A) PI3K in controlling heart growth and to circumvent potential complications from the overexpression of dominant negative and constitutively active proteins, we generated mice with muscle-specific deletion of the p85alpha regulatory subunit and germ line deletion of the p85beta regulatory subunit of class I(A) PI3K. Here we show that mice with cardiac deletion of both p85 subunits exhibit attenuated Akt signaling in the heart, reduced heart size, and altered cardiac gene expression. Furthermore, exercise-induced cardiac hypertrophy is also attenuated in the p85 knockout hearts. Despite such defects in postnatal developmental growth and physiological hypertrophy, the p85 knockout hearts exhibit normal contractility and myocardial histology. Our results therefore provide strong genetic evidence that class I(A) PI3Ks are critical regulators for the developmental growth and physiological hypertrophy of the heart.

  13. Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta Has an Essential Function in Cell Survival ▿

    PubMed Central

    Kumar, Amit; Redondo-Muñoz, Javier; Perez-García, Vicente; Cortes, Isabel; Chagoyen, Monica; Carrera, Ana C.

    2011-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival. PMID:21383062

  14. Phosphoinositide 3-kinase beta controls replication factor C assembly and function

    PubMed Central

    Redondo-Muñoz, Javier; Josefa Rodríguez, María; Silió, Virginia; Pérez-García, Vicente; María Valpuesta, José; Carrera, Ana C.

    2013-01-01

    Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1– and RFC–RAD17 complexes, but also RFC–CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes. PMID:23175608

  15. Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

    PubMed Central

    Pérez-García, Vicente; Redondo-Muñoz, Javier; Kumar, Amit

    2014-01-01

    The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P3 levels. PMID:24958106

  16. Phosphoinositide 3-Kinase Beta Protects Nuclear Envelope Integrity by Controlling RCC1 Localization and Ran Activity

    PubMed Central

    Redondo-Muñoz, Javier; Pérez-García, Vicente; Rodríguez, María J.; Valpuesta, José M.

    2014-01-01

    The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran. PMID:25348717

  17. Creatine inhibits adipogenesis by downregulating insulin-induced activation of the phosphatidylinositol 3-kinase signaling pathway.

    PubMed

    Lee, Nayeon; Kim, Inhee; Park, Soojeong; Han, Dasol; Ha, Soobong; Kwon, Mookwang; Kim, Juwan; Byun, Sung-Hyun; Oh, Wonil; Jeon, Hong Bae; Kweon, Dae-Hyuk; Cho, Jae Youl; Yoon, Keejung

    2015-04-15

    Creatine is a nitrogenous organic acid known to function in adenosine triphosphate (ATP) metabolism. Recent evidence indicates that creatine regulates the differentiation of mesenchymal stem cells (MSCs) in processes such as osteogenesis and myogenesis. In this study, we show that creatine also has a negative regulatory effect on fat cell formation. Creatine inhibits the accumulation of cytoplasmic triglycerides in a dose-dependent manner irrespective of the adipogenic cell models used, including a C3H10T1/2 MSC line, 3T3-L1 preadipocytes, and primary human MSCs. Consistently, a dramatic reduction in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), glucose transporters, 1 and 4 (Glut1, Glut4), and adipocyte markers, aP2 and adipsin, was observed in the presence of creatine. Creatine appears to exert its inhibitory effects on adipogenesis during early differentiation, but not late differentiation, or proliferation stages through inhibition of the PI3K-Akt-PPARγ signaling pathway. In an in vivo model, administration of creatine into mice resulted in body mass increase without fat accumulation. In summary, our results indicate that creatine downregulates adipogenesis through inhibition of phosphatidylinositol 3-kinase (PI3K) activation and imply the potent therapeutic value of creatine in treating obesity and obesity-related metabolic disorders.

  18. Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms.

    PubMed

    Kaneko, Kazuma; Ueki, Kohjiro; Takahashi, Noriko; Hashimoto, Shinji; Okamoto, Masayuki; Awazawa, Motoharu; Okazaki, Yukiko; Ohsugi, Mitsuru; Inabe, Kazunori; Umehara, Toshihiro; Yoshida, Masashi; Kakei, Masafumi; Kitamura, Tadahiro; Luo, Ji; Kulkarni, Rohit N; Kahn, C Ronald; Kasai, Haruo; Cantley, Lewis C; Kadowaki, Takashi

    2010-12-01

    Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.

  19. Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

    PubMed Central

    Young, Lindsey N.; Cho, Kelvin; Lawrence, Rosalie; Zoncu, Roberto; Hurley, James H.

    2016-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure. PMID:27385829

  20. Biliverdin Reductase Mediates Hypoxia-Induced EMT via PI3-Kinase and Akt

    PubMed Central

    Zeng, Rui; Yao, Ying; Han, Min; Zhao, Xiaoqin; Liu, Xiao-Cheng; Wei, Juncheng; Luo, Yun; Zhang, Juan; Zhou, Jianfeng; Wang, Shixuan; Ma, Ding; Xu, Gang

    2008-01-01

    Chronic hypoxia in the renal parenchyma is thought to induce epithelial-to-mesenchymal transition (EMT), leading to fibrogenesis and ultimately end-stage renal failure. Biliverdin reductase, recently identified as a serine/threonine/tyrosine kinase that may activate phosphatidylinositol 3-kinase (PI3K) and Akt, is upregulated in response to reactive oxygen species that may accompany hypoxia. We investigated this potential role of biliverdin reductase in hypoxia-induced renal tubular EMT. Expression of biliverdin reductase was upregulated in a human proximal tubule cell line (HK-2) cultured in hypoxic conditions (1% O2), and this was accompanied by reduced expression of E-cadherin and increased expression of the mesenchymal marker vimentin. Inhibiting PI3K reversed these changes, consistent with EMT. In normoxic conditions, overexpression of biliverdin reductase promoted similar characteristics of EMT, which were also reversed by inhibiting PI3K. Furthermore, using small interfering RNA (siRNA) to knockdown biliverdin reductase, we demonstrated that the enzyme associates with phosphorylated Akt and mediates the hypoxia-induced EMT phenotype. In vivo, expression of biliverdin reductase increased in the tubular epithelia of 5/6-nephrectomized rats, and immunohistochemistry of serial sections demonstrated similar localization of phosphorylated Akt and biliverdin reductase. In conclusion, biliverdin reductase mediates hypoxia-induced EMT through a PI3K/Akt-dependent pathway. PMID:18184861

  1. Targeting phosphoinositide 3-kinase δ for the treatment of respiratory diseases.

    PubMed

    Sriskantharajah, Srividya; Hamblin, Nicole; Worsley, Sally; Calver, Andrew R; Hessel, Edith M; Amour, Augustin

    2013-03-01

    Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3Kδ may restore steroid effectiveness under conditions of oxidative stress. PI3Kδ inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3Kδ pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3Kδ for the treatment of respiratory disease, focusing on recent developments in the role of the PI3Kδ pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD.

  2. Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk

    PubMed Central

    Stevens, K N; Garcia-Closas, M; Fredericksen, Z; Kosel, M; Pankratz, V S; Hopper, J L; Dite, G S; Apicella, C; Southey, M C; Schmidt, M K; Broeks, A; Van ‘t Veer, L J; Tollenaar, R A E M; Fasching, P A; Beckmann, M W; Hein, A; Ekici, A B; Johnson, N; Peto, J; dos Santos Silva, I; Gibson, L; Sawyer, E; Tomlinson, I; Kerin, M J; Chanock, S; Lissowska, J; Hunter, D J; Hoover, R N; Thomas, G D; Milne, R L; Pérez, JI Arias; González-Neira, A; Benítez, J; Burwinkel, B; Meindl, A; Schmutzler, R K; Bartrar, C R; Hamann, U; Ko, Y D; Brüning, T; Chang-Claude, J; Hein, R; Wang-Gohrke, S; Dörk, T; Schürmann, P; Bremer, M; Hillemanns, P; Bogdanova, N; Zalutsky, J V; Rogov, Y I; Antonenkova, N; Lindblom, A; Margolin, S; Mannermaa, A; Kataja, V; Kosma, V-M; Hartikainen, J; Chenevix-Trench, G; Chen, X; Peterlongo, P; Bonanni, B; Bernard, L; Manoukian, S; Wang, X; Cerhan, J; Vachon, C M; Olson, J; Giles, G G; Baglietto, L; McLean, C A; Severi, G; John, E M; Miron, A; Winqvist, R; Pylkäs, K; Jukkola-Vuorinen, A; Grip, M; Andrulis, I; Knight, J A; Glendon, G; Mulligan, A M; Cox, A; Brock, I W; Elliott, G; Cross, S S; Pharoah, P P; Dunning, A M; Pooley, K A; Humphreys, M K; Wang, J; Kang, D; Yoo, K-Y; Noh, D-Y; Sangrajrang, S; Gabrieau, V; Brennan, P; McKay, J; Anton-Culver, H; Ziogas, A; Couch, F J; Easton, D F

    2011-01-01

    Background: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. Methods: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). Results: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95–0.99, P=4.6 × 10−3), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96–1.01, P=0.139). Conclusion: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer PMID:22033276

  3. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    SciTech Connect

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori . E-mail: hirokato@pharm.kyoto-u.ac.jp

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.

  4. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

    PubMed

    Linassier, C; MacDougall, L K; Domin, J; Waterfield, M D

    1997-02-01

    Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

  5. Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

    PubMed

    Yum, H K; Arcaroli, J; Kupfner, J; Shenkar, R; Penninger, J M; Sasaki, T; Yang, K Y; Park, J S; Abraham, E

    2001-12-01

    Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.

  6. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers

    PubMed Central

    Geering, Barbara; Cutillas, Pedro R.; Nock, Gemma; Gharbi, Severine I.; Vanhaesebroeck, Bart

    2007-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110α, p110β, or p110δ) bound to any of five distinct regulatory subunits (p85α, p85β, p55γ, p55α, and p50α, collectively referred to as “p85s”). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85α has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000–15,000 p85/p110 complexes per cell, with p110β and p110δ being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms. PMID:17470792

  7. Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

    PubMed

    Carter, Edward; Miron-Buchacra, Gabriela; Goldoni, Silvia; Danahay, Henry; Westwick, John; Watson, Malcolm L; Tosh, David; Ward, Stephen G

    2014-01-01

    Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.

  8. Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells

    PubMed Central

    Oppermann, Sina; Lam, Avery J.; Tung, Stephanie; Shi, Yonghong; McCaw, Lindsay; Wang, Guizhei; Ylanko, Jarkko; Leber, Brian; Andrews, David; Spaner, David E.

    2016-01-01

    Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors. PMID:27579615

  9. Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

    SciTech Connect

    Balajee, A.S.; Meador, J.A.; Su, Y.

    2011-03-24

    It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellular mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the

  10. A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation.

    PubMed

    Franco-Echevarría, Elsa; Baños-Sanz, Jose I; Monterroso, Begoña; Round, Adam; Sanz-Aparicio, Julia; González, Beatriz

    2014-11-01

    IP3-3K [Ins(1,4,5)P3 3-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with Ins(1,3,4,5)P4 regulates Ca2+ homoeostasis. In addition, Ins(1,3,4,5)P4 is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.

  11. Role of class III phosphatidylinositol 3-kinase during programmed nuclear death of Tetrahymena thermophila.

    PubMed

    Akematsu, Takahiko; Fukuda, Yasuhiro; Attiq, Rizwan; Pearlman, Ronald E

    2014-02-01

    Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program.

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

    SciTech Connect

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

    2014-07-18

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

  13. Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides.

    PubMed

    Laval, S; Laklai, H; Fanjul, M; Pucelle, M; Laurell, H; Billon-Galés, A; Le Guellec, S; Delisle, M-B; Sonnenberg, A; Susini, C; Pyronnet, S; Bousquet, C

    2014-04-10

    Given the failure of chemo- and biotherapies to fight advanced pancreatic cancer, one major challenge is to identify critical events that initiate invasion. One priming step in epithelia carcinogenesis is the disruption of epithelial cell anchorage to the basement membrane which can be provided by hemidesmosomes (HDs). However, the existence of HDs in pancreatic ductal epithelium and their role in carcinogenesis remain unexplored. HDs have been explored in normal and cancer pancreatic cells, and patient samples. Unique cancer cell models where HD assembly can be pharmacologically manipulated by somatostatin/sst2 signaling have been then used to investigate the role and molecular mechanisms of dynamic HD during pancreatic carcinogenesis. We surprisingly report the presence of mature type-1 HDs comprising the integrin α6β4 and bullous pemphigoid antigen BP180 in the human pancreatic ductal epithelium. Importantly, HDs are shown to disassemble during pancreatic carcinogenesis. HD breakdown requires phosphoinositide 3-kinase (PI3K)-dependent induction of the matrix-metalloprotease MMP-9, which cleaves BP180. Consequently, integrin α6β4 delocalizes to the cell-leading edges where it paradoxically promotes cell migration and invasion through S100A4 activation. As S100A4 in turn stimulates MMP-9 expression, a vicious cycle maintains BP180 cleavage. Inactivation of this PI3K-MMP-9-S100A4 signaling loop conversely blocks BP180 cleavage, induces HD reassembly and inhibits cell invasion. We conclude that mature type-1 HDs are critical anchoring structures for the pancreatic ductal epithelium whose disruption, upon PI3K activation during carcinogenesis, provokes pancreatic cancer cell migration and invasion.

  14. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    PubMed Central

    Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

    2014-01-01

    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity. PMID:27919038

  15. Phosphoinositide 3-kinase p110δ mediates estrogen- and FSH-stimulated ovarian follicle growth.

    PubMed

    Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing; Fan, Heng-Yu; Sha, Jiahao

    2013-09-01

    In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development.

  16. The Association of PI3 Kinase Signaling and Chemoresistance in Advanced Ovarian Cancer

    PubMed Central

    Carden, Craig P.; Stewart, Adam; Thavasu, Parames; Kipps, Emma; Pope, Lorna; Crespo, Mateus; Miranda, Susana; Attard, Gerhardt; Garrett, Michelle D.; Clarke, Paul A.; Workman, Paul; de Bono, Johann S.; Gore, Martin; Kaye, Stan B; Banerji, Udai

    2015-01-01

    Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN. PMID:22556379

  17. Enterococcus faecalis infection activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2014-12-01

    Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.

  18. Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1

    PubMed Central

    Otero, Miguel; Lago, Rocío; Lago, Francisca; Reino, Juan Jesús Gomez; Gualillo, Oreste

    2005-01-01

    The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and IL-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 μmol/l] and LY294002 [1, 2.5, 5 and 10 μmol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 μmol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 μmol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with IL-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and IL-1 resulted in a net increase in nitric oxide concentration over IL-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/IL-1. Finally, wortmannin, LY294002, PD098059 and SB203580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5 chondrocytes, and

  19. Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1.

    PubMed

    Otero, Miguel; Lago, Rocío; Lago, Francisca; Reino, Juan Jesús Gomez; Gualillo, Oreste

    2005-01-01

    The objective of the present study was to investigate the effect of leptin, alone or in combination with IL-1, on nitric oxide synthase (NOS) type II activity in vitro in human primary chondrocytes, in the mouse chondrogenic ATDC5 cell line, and in mature and hypertrophic ATDC5 differentiated chondrocytes. For completeness, we also investigated the signalling pathway of the putative synergism between leptin and IL-1. For this purpose, nitric oxide production was evaluated using the Griess colorimetric reaction in culture medium of cells stimulated over 48 hours with leptin (800 nmol/l) and IL-1 (0.025 ng/ml), alone or combined. Specific pharmacological inhibitors of NOS type II (aminoguanidine [1 mmol/l]), janus kinase (JAK)2 (tyrphostin AG490 and Tkip), phosphatidylinositol 3-kinase (PI3K; wortmannin [1, 2.5, 5 and 10 micromol/l] and LY294002 [1, 2.5, 5 and 10 micromol/l]), mitogen-activated protein kinase kinase (MEK)1 (PD098059 [1, 5, 10, 20 and 30 micromol/l]) and p38 kinase (SB203580 [1, 5, 10, 20 and 30 micromol/l]) were added 1 hour before stimulation. Nitric oxide synthase type II mRNA expression in ATDC5 chondrocytes was investigated by real-time PCR and NOS II protein expression was analyzed by western blot. Our results indicate that stimulation of chondrocytes with IL-1 results in dose-dependent nitric oxide production. In contrast, leptin alone was unable to induce nitric oxide production or expression of NOS type II mRNA or its protein. However, co-stimulation with leptin and IL-1 resulted in a net increase in nitric oxide concentration over IL-1 challenge that was eliminated by pretreatment with the NOS II specific inhibitor aminoguanidine. Pretreatment with tyrphostin AG490 and Tkip (a SOCS-1 mimetic peptide that inhibits JAK2) blocked nitric oxide production induced by leptin/IL-1. Finally, wortmannin, LY294002, PD098059 and SB203580 significantly decreased nitric oxide production. These findings were confirmed in mature and hypertrophic ATDC5

  20. Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: A crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

    SciTech Connect

    Bernard, Laurence; Legay, Christine; Adriaenssens, Eric; Mougel, Alexandra; Ricort, Jean-Marc . E-mail: ricort@lbpa.ens-cachan.fr

    2006-12-01

    Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110 expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.

  1. Glabridin induces glucose uptake via the AMP-activated protein kinase pathway in muscle cells.

    PubMed

    Sawada, Keisuke; Yamashita, Yoko; Zhang, Tianshun; Nakagawa, Kaku; Ashida, Hitoshi

    2014-08-05

    The present study demonstrates that glabridin, a prenylated isoflavone in licorice, stimulates glucose uptake through the adenosine monophosphate-activated protein kinase (AMPK) pathway in L6 myotubes. Treatment with glabridin for 4h induced glucose uptake in a dose-dependent manner accompanied by the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane. Glabridin needed at least 4h to increase glucose uptake, while it significantly decreased glycogen and increased lactic acid within 15 min. Pharmacological inhibition of AMPK by Compound C suppressed the glabridin-induced glucose uptake, whereas phosphoinositide 3-kinase and Akt inhibition by LY294002 and Akt1/2 inhibitor, respectively, did not. Furthermore, glabridin induced AMPK phosphorylation, and siRNA for AMPK completely abolished glabridin-induced glucose uptake. We confirmed that glabridin-rich licorice extract prevent glucose intolerance accompanied by the AMPK-dependent GLUT4 translocation in the plasma membrane of mice skeletal muscle. These results indicate that glabridin may possess a therapeutic effect on metabolic disorders, such as diabetes and hyperglycemia, by modulating glucose metabolism through AMPK in skeletal muscle cells.

  2. PPARβ/δ, a Novel Regulator for Vascular Smooth Muscle Cells Phenotypic Modulation and Vascular Remodeling after Subarachnoid Hemorrhage in Rats.

    PubMed

    Zhang, Hongrong; Jiang, Li; Guo, Zongduo; Zhong, Jianjun; Wu, Jingchuan; He, Junchi; Liu, Han; He, Zhaohui; Wu, Haitao; Cheng, Chongjie; Sun, Xiaochuan

    2017-03-22

    Cerebral vascular smooth muscle cell (VSMC) phenotypic switch is involved in the pathophysiology of vascular injury after aneurysmal subarachnoid hemorrhage (aSAH), whereas the molecular mechanism underlying it remains largely speculative. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been implicated to modulate the vascular cells proliferation and vascular homeostasis. In the present study, we investigated the potential role of PPARβ/δ in VSMC phenotypic switch following SAH. Activation of PPARβ/δ by GW0742 and adenoviruses PPARβ/δ (Ad-PPARβ/δ) significantly inhibited hemoglobin-induced VSMC phenotypic switch. However, the effects of PPARβ/δ on VSMC phenotypic switch were partly obstacled in the presence of LY294002, a potent inhibitor of Phosphatidyl-Inositol-3 Kinase-AKT (PI3K/AKT). Furthermore, following study demonstrated that PPARβ/δ-induced PI3K/AKT activation can also contribute to Serum Response Factor (SRF) nucleus localization and Myocardin expression, which was highly associated with VSMC phenotypic switch. Finally, we found that Ad-PPARβ/δ positively modulated vascular remodeling in SAH rats, i.e. the diameter of basilar artery and the thickness of vessel wall. In addition, overexpression of PPARβ/δ by adenoviruses significantly improved neurological outcome. Taken together, this study identified PPARβ/δ as a useful regulator for VSMC phenotypic switch and vascular remodeling following SAH, providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

  3. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

    PubMed

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-11-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

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

    PubMed

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

    2015-04-28

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

  5. Acetyl-l-carnitine restores synaptic transmission and enhances the inducibility of stable LTP after oxygen-glucose deprivation.

    PubMed

    Kocsis, Kitti; Frank, Rita; Szabó, József; Knapp, Levente; Kis, Zsolt; Farkas, Tamás; Vécsei, László; Toldi, József

    2016-09-22

    Hypoxic circumstances result in functional and structural impairments of the brain. Oxygen-glucose deprivation (OGD) on hippocampal slices is a technique widely used to investigate the consequences of ischemic stroke and the potential neuroprotective effects of different drugs. Acetyl-l-carnitine (ALC) is a naturally occurring substance in the body, and it can therefore be administered safely even in relatively high doses. In previous experiments, ALC pretreatment proved to be effective against global hypoperfusion. In the present study, we investigated whether ALC can be protective in an OGD model. We are not aware of any earlier study in which the long-term potentiation (LTP) function on hippocampal slices was measured after OGD. Therefore, we set out to determine whether an effective ALC concentration has an effect on synaptic plasticity after OGD in the hippocampal CA1 subfield of rats. A further aim was to investigate the mechanism underlying the protective effect of this compound. The experiments revealed that ALC is neuroprotective against OGD in a dose-dependent manner, which is manifested not only in the regeneration of the impaired synaptic transmission after the OGD, but also in the inducibility and stability of the LTP. In the case of the most effective concentration of ALC (500μM), use of a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) revealed that the PI3K/Akt signaling pathway has a key role in the restoration of the synaptic transmission and plasticity reached by ALC treatment.

  6. Rapid-acting antidepressant-like effects of acetyl-l-carnitine mediated by PI3K/AKT/BDNF/VGF signaling pathway in mice.

    PubMed

    Wang, W; Lu, Y; Xue, Z; Li, C; Wang, C; Zhao, X; Zhang, J; Wei, X; Chen, X; Cui, W; Wang, Q; Zhou, W

    2015-01-29

    The possible involvement of the PI3K/AKT/BDNF/VGF signaling in rapid-acting antidepressant-like effects of antidepressants has been explored progressively by more studies. However, whether this signaling participates in the antidepressant-like effects of acetyl-l-carnitine (ALC) has not been examined. Herein, we assessed the antidepressant-like effects of ALC using the forced swimming test (FST). Our results demonstrated the dose-effect relationship of acute administration of ALC (5, 25, 50 and 100mg/kg, i.p.) and showed that it dose-dependently decreased the immobility time on FST of mice. In addition, ALC (100 mg/kg, i.p.) also reversed depressive-like behavior and the down-regulation of phosphorylated AKT (pAKT), brain-derived neurotrophic factor (BDNF) and neuropeptide VGF in the hippocampus and prefrontal cortex of mice induced by chronic unpredictable mild stress (CUMS) paradigm. Further, intra-cerebroventricular (i.c.v.) infusions of LY294002 (10 nmol/side), a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly prevented the antidepressant-like effect of ALC (100mg/kg, i.p.). In conclusion, our results demonstrated that ALC exerts rapid-acting antidepressant-like effects that might be mediated by the PI3K/AKT/BDNF/VGF signaling pathway.

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

    PubMed Central

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

    2016-01-01

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

  8. Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

    SciTech Connect

    Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun . E-mail: dli2@slu.edu

    2006-11-01

    Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), {delta}p85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.

  9. Both Creatine and Its Product Phosphocreatine Reduce Oxidative Stress and Afford Neuroprotection in an In Vitro Parkinson’s Model

    PubMed Central

    Martín-de-Saavedra, Maria D.; Romero, Alejandro; Egea, Javier; Ludka, Fabiana K.; Tasca, Carla I.; Farina, Marcelo; Rodrigues, Ana Lúcia S.; López, Manuela G.

    2014-01-01

    Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA)-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson’s model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK3β) was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine473) and GSK3β (Serine9). Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons. PMID:25424428

  10. Both creatine and its product phosphocreatine reduce oxidative stress and afford neuroprotection in an in vitro Parkinson's model.

    PubMed

    Cunha, Mauricio Peña; Martín-de-Saavedra, Maria D; Romero, Alejandro; Egea, Javier; Ludka, Fabiana K; Tasca, Carla I; Farina, Marcelo; Rodrigues, Ana Lúcia S; López, Manuela G

    2014-01-01

    Creatine is the substrate for creatine kinase in the synthesis of phosphocreatine (PCr). This energetic system is endowed of antioxidant and neuroprotective properties and plays a pivotal role in brain energy homeostasis. The purpose of this study was to investigate the neuroprotective effect of creatine and PCr against 6-hydroxydopamine (6-OHDA)-induced mitochondrial dysfunction and cell death in rat striatal slices, used as an in vitro Parkinson's model. The possible involvement of the signaling pathway mediated by phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK3β) was also evaluated. Exposure of striatal slices to 6-OHDA caused a significant disruption of the cellular homeostasis measured as 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction, lactate dehydrogenase release, and tyrosine hydroxylase levels. 6-OHDA exposure increased the levels of reactive oxygen species and thiobarbituric acid reactive substances production and decreased mitochondrial membrane potential in rat striatal slices. Furthermore, 6-OHDA decreased the phosphorylation of Akt (Serine(473)) and GSK3β (Serine(9)). Coincubation with 6-OHDA and creatine or PCr reduced the effects of 6-OHDA toxicity. The protective effect afforded by creatine or PCr against 6-OHDA-induced toxicity was reversed by the PI3K inhibitor LY294002. In conclusion, creatine and PCr minimize oxidative stress in striatum to afford neuroprotection of dopaminergic neurons.

  11. Insulin-like growth factor 1 specifically up-regulates expression of modifier subunit of glutamate-cysteine ligase and enhances glutathione synthesis in SH-SY5Y cells.

    PubMed

    Takahashi, Shuhei; Hisatsune, Akinori; Kurauchi, Yuki; Seki, Takahiro; Katsuki, Hiroshi

    2016-01-15

    Glutathione is a key regulator of oxidative balance in all mammals, especially in the central nervous system. The first step of glutathione synthesis is catalyzed by glutamate-cysteine ligase (GCL), which is composed of catalytic and modifier subunits (GCLC and GCLM, respectively). In non-neural cells and tissues, insulin and insulin-like growth factor 1 (IGF-1) have been found to stimulate transcription of GCLC gene. Here we found that treatment of human neuroblastoma SH-SY5Y cells with insulin or IGF-1 increased mRNA level of GCLM, but not of GCLC, in a concentration- and time-dependent manner. In contrast, insulin did not increase GCL expression in rat C6 glioma cells. We also confirmed that IGF-1 increased protein level of GCLM and cellular glutathione content in SH-SY5Y cells. In addition, IGF-1 increased nuclear factor erythroid 2-related factor 2 (Nrf2) protein in the nuclear fraction of SH-SY5Y cells. siRNA-mediated knockdown of Nrf2 protein expression abrogated IGF-1-induced up-regulation of GCLM mRNA expression. Finally, IGF-1-induced increase in nuclear Nrf2 protein and GCLM mRNA expression was abolished by LY294002, a phosphoinositide 3-kinase inhibitor. These results indicate that insulin and IGF-1 have the ability to enhance glutathione biosynthesis in neuronal cells via specific up-regulation of GCLM expression.

  12. A novel compound DSC suppresses lipopolysaccharide-induced inflammatory responses by inhibition of Akt/NF-κB signalling in macrophages.

    PubMed

    Liu, Xin-Hua; Pan, Li-Long; Jia, Yao-Ling; Wu, Dan; Xiong, Qing-Hui; Wang, Yang; Zhu, Yi-Zhun

    2013-05-15

    A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2-ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)], derived from Danshensu, exerted cytoprotective effects by anti-oxidative and anti-apoptotic activities in vitro. Herein, we reported the protective effects of DSC on lipopolysaccharide (LPS)-induced inflammatory responses in murine RAW264.7 macrophages and the underlying mechanisms. We showed that DSC concentration-dependently attenuated nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression with less cytotoxicity. Signal transduction studies indicated that DSC significantly inhibited LPS-induced phosphorylation of Akt, but not c-Jun N-terminal kinase 1/2, p38, or extracellular signal-regulated kinase 1/2. Meanwhile, LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) p65 was decreased by DSC. Furthermore, a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 significantly suppressed LPS-induced NF-κB p65 nuclear translocation, iNOS expression, and NO production, which was also mimicked by pretreatment with DSC. These results suggested that DSC attenuated LPS-induced inflammatory response in macrophages, at least in part, through suppression of PI3K/Akt signaling and NF-κB activation.

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2014-05-01

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

  15. Ethanol stimulates epithelial sodium channels by elevating reactive oxygen species.

    PubMed

    Bao, Hui-Fang; Song, John Z; Duke, Billie J; Ma, He-Ping; Denson, Donald D; Eaton, Douglas C

    2012-12-01

    Alcohol affects total body sodium balance, but the molecular mechanism of its effect remains unclear. We used single-channel methods to examine how ethanol affects epithelial sodium channels (ENaC) in A6 distal nephron cells. The data showed that ethanol significantly increased both ENaC open probability (P(o)) and the number of active ENaC in patches (N). 1-Propanol and 1-butanol also increased ENaC activity, but iso-alcohols did not. The effects of ethanol were mimicked by acetaldehyde, the first metabolic product of ethanol, but not by acetone, the metabolic product of 2-propanol. Besides increasing open probability and apparent density of active channels, confocal microscopy and surface biotinylation showed that ethanol significantly increased α-ENaC protein in the apical membrane. The effects of ethanol on ENaC P(o) and N were abolished by a superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) and blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Consistent with an effect of ethanol-induced reactive oxygen species (ROS) on ENaC, primary alcohols and acetaldehyde elevated intracellular ROS, but secondary alcohols did not. Taken together with our previous finding that ROS stimulate ENaC, the current results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde.

  16. PPARβ/δ, a Novel Regulator for Vascular Smooth Muscle Cells Phenotypic Modulation and Vascular Remodeling after Subarachnoid Hemorrhage in Rats

    PubMed Central

    Zhang, Hongrong; Jiang, Li; Guo, Zongduo; Zhong, Jianjun; Wu, Jingchuan; He, Junchi; Liu, Han; He, Zhaohui; Wu, Haitao; Cheng, Chongjie; Sun, Xiaochuan

    2017-01-01

    Cerebral vascular smooth muscle cell (VSMC) phenotypic switch is involved in the pathophysiology of vascular injury after aneurysmal subarachnoid hemorrhage (aSAH), whereas the molecular mechanism underlying it remains largely speculative. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been implicated to modulate the vascular cells proliferation and vascular homeostasis. In the present study, we investigated the potential role of PPARβ/δ in VSMC phenotypic switch following SAH. Activation of PPARβ/δ by GW0742 and adenoviruses PPARβ/δ (Ad-PPARβ/δ) significantly inhibited hemoglobin-induced VSMC phenotypic switch. However, the effects of PPARβ/δ on VSMC phenotypic switch were partly obstacled in the presence of LY294002, a potent inhibitor of Phosphatidyl-Inositol-3 Kinase-AKT (PI3K/AKT). Furthermore, following study demonstrated that PPARβ/δ-induced PI3K/AKT activation can also contribute to Serum Response Factor (SRF) nucleus localization and Myocardin expression, which was highly associated with VSMC phenotypic switch. Finally, we found that Ad-PPARβ/δ positively modulated vascular remodeling in SAH rats, i.e. the diameter of basilar artery and the thickness of vessel wall. In addition, overexpression of PPARβ/δ by adenoviruses significantly improved neurological outcome. Taken together, this study identified PPARβ/δ as a useful regulator for VSMC phenotypic switch and vascular remodeling following SAH, providing novel insights into the therapeutic strategies of delayed cerebral ischemia. PMID:28327554

  17. Biological Effect of Licochalcone C on the Regulation of PI3K/Akt/eNOS and NF-κB/iNOS/NO Signaling Pathways in H9c2 Cells in Response to LPS Stimulation.

    PubMed

    Franceschelli, Sara; Pesce, Mirko; Ferrone, Alessio; Gatta, Daniela Maria Pia; Patruno, Antonia; Lutiis, Maria Anna De; Quiles, José Luis; Grilli, Alfredo; Felaco, Mario; Speranza, Lorenza

    2017-03-23

    Polyphenols compounds are a group molecules present in many plants. They have antioxidant properties and can also be helpful in the management of sepsis. Licochalcone C (LicoC), a constituent of Glycyrrhiza glabra, has various biological and pharmacological properties. In saying this, the effect of LicoC on the inflammatory response that characterizes septic myocardial dysfunction is poorly understood. The aim of this study was to determine whether LicoC exhibits anti-inflammatory properties on H9c2 cells that are stimulated with lipopolysaccharide. Our results have shown that LicoC treatment represses nuclear factor-κB (NF-κB) translocation and several downstream molecules, such as inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, LicoC has upregulated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signaling pathway. Finally, 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), a specific PI3K inhibitor, blocked the protective effects of LicoC. These findings indicate that LicoC plays a pivotal role in cardiac dysfunction in sepsis-induced inflammation.

  18. Carbocisteine reduces virus-induced pulmonary inflammation in mice exposed to cigarette smoke.

    PubMed

    Yageta, Yuichi; Ishii, Yukio; Morishima, Yuko; Ano, Satoshi; Ohtsuka, Shigeo; Matsuyama, Masashi; Takeuchi, Kaoru; Itoh, Ken; Yamamoto, Masayuki; Hizawa, Nobuyuki

    2014-05-01

    Carbocisteine (S-CMC) inhibits viral infection and prevents acute exacerbation of chronic obstructive pulmonary disease. We recently demonstrated the protective effects of NF-E2-related factor (Nrf) 2 against influenza virus (FluV)-induced pulmonary inflammation in mice exposed to cigarette smoke (CS). In our current study, we investigated the effects of S-CMC on Nrf2 activation in cultured macrophages, and in mice infected with influenza after exposure to CS. Nuclear translocation of Nrf2 and the expression of Nrf2-targeted antioxidant genes, such as heavy and light subunits of γ glutamyl cysteine synthetase and heme oxigenase-1, were enhanced in a dose-dependent manner after treatment with S-CMC in peritoneal and alveolar macrophages of wild-type mice, but not in those of Nrf2-deficient mice. Nuclear translocation of Nrf2 in macrophages was inhibited by the phosphatidylinositol 3-kinase inhibitor, LY294002. Phosphorylated Akt, Nrf2, and heme oxigenase-1 were induced in the alveolar macrophages of the lungs in wild-type mice after S-CMC administration. The extent of oxidative stress, inflammatory cell infiltration, pulmonary edema, and goblet cell hyperplasia was suppressed by S-CMC administration in the lungs of wild-type mice after exposure to both CS and FluV. Our findings suggest that S-CMC reduces pulmonary inflammation and mucus overproduction in mice exposed to CS after infection with FluV via the activation of Nrf2.

  19. Piperine, a dietary phytochemical, inhibits angiogenesis

    PubMed Central

    Doucette, Carolyn D.; Hilchie, Ashley L.; Liwski, Robert; Hoskin, David W.

    2012-01-01

    Angiogenesis plays an important role in tumor progression. Piperine, a major alkaloid constituent of black pepper, has diverse physiological actions including killing of cancer cells; however, the effect of piperine on angiogenesis is not known. Here we show that piperine inhibited the proliferation and G1/S transition of human umbilical vein endothelial cells (HUVECs) without causing cell death. Piperine also inhibited HUVEC migration and tubule formation in vitro, as well as collagen-induced angiogenic activity by rat aorta explants and breast cancer cell-induced angiogenesis in chick embryos. Although piperine binds to and activates the cation channel transient receptor potential vanilloid 1 (TRPV1), its effects on endothelial cells did not involve TRPV1 since the antiproliferative effect of piperine was not affected by TRPV1-selective antagonists, nor did HUVECs express detectable TRPV1 mRNA. Importantly, piperine inhibited phosphorylation of Ser 473 and Thr 308 residues of Akt (protein kinase B), which is a key regulator of endothelial cell function and angiogenesis. Consistent with Akt inhibition as the basis of piperine’s action on HUVECs, inhibition of the phosphoinositide-3 kinase/Akt signaling pathway with LY-294002 also inhibited HUVEC proliferation and collagen-induced angiogenesis. Taken together, these data support the further investigation of piperine as an angiogenesis inhibitor for use in cancer treatment. PMID:22902327

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

    PubMed

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

    2012-11-01

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

  1. Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells.

    PubMed

    Zhou, Yuan; He, Yongzheng; Sharma, Richa; Xing, Wen; Estwick, Selina A; Wu, Xiaohua; Rhodes, Steven D; Xu, Mingjiang; Yang, Feng-Chun

    2015-06-01

    Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

  2. Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells.

    PubMed

    Tsai, Jen-Pi; Lee, Chien-Hsing; Ying, Tsung-Ho; Lin, Chu-Liang; Lin, Chia-Liang; Hsueh, Jung-Tsung; Hsieh, Yi-Hsien

    2015-10-06

    The use of dietary bioactive compounds in chemoprevention can potentially reverse, suppress, or even prevent cancer progression. However, the effects of licochalcone A (LicA) on apoptosis and autophagy in cervical cancer cells have not yet been clearly elucidated. In this study, LicA treatment was found to significantly induce the apoptotic and autophagic capacities of cervical cancer cells in vitro and in vivo. MTT assay results showed dose- and time-dependent cytotoxicity in four cervical cancer cell lines treated with LicA. We found that LicA induced mitochondria-dependent apoptosis in SiHa cells, with decreasing Bcl-2 expression. LicA also induced autophagy effects were examined by identifying accumulation of Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3)-II. Treatment with autophagy-specific inhibitors (3-methyladenine and bafilomycin A1) enhanced LicA-induced apoptosis. In addition, we suggested the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of mTOR pathway by LicA. Furthermore, the inhibition of PI3K/Akt by LY294002/si-Akt or of mTOR by rapamycin augmented LicA-induced apoptosis and autophagy. Finally, the in vivo mice bearing a SiHa xenograft, LicA dosed at 10 or 20 mg/kg significantly inhibited tumor growth. Our findings demonstrate the chemotherapeutic potential of LicA for treatment of human cervical cancer.

  3. Bilirubin exerts pro-angiogenic property through Akt-eNOS-dependent pathway.

    PubMed

    Ikeda, Yasumasa; Hamano, Hirofumi; Satoh, Akiho; Horinouchi, Yuya; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Ishizawa, Keisuke; Aihara, Ken-Ichi; Tsuchiya, Koichiro; Tamaki, Toshiaki

    2015-11-01

    Low serum bilirubin levels are associated with the risk of cardiovascular diseases including peripheral artery disease. Bilirubin is known to exert its property such as antioxidant effect or the enhancement of flow-mediated vasodilation, however, bilirubin action on angiogenesis remains unclear. To investigate the molecular mechanism of bilirubin on angiogenic effect, we first employed C57BL/6J mice with unilateral hindlimb ischemia surgery and divided the mice into two groups (vehicle-treated group and bilirubin-treated group). The analysis of laser speckle blood flow demonstrated the enhancement of blood flow recovery in response to ischemia of mice with bilirubin treatment. The density of capillaries was significantly higher in ischemic-adductor muscles of bilirubin-treated mice. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were increased in ischemic skeletal muscles of mice with bilirubin treatment compared with vehicle treatment. In in vitro experiments by using human aortic endothelial cells, bilirubin augmented eNOS and Akt phosphorylation, cell proliferation, cell migration and tube formation. These bilirubin actions on endothelial cell activation were inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor. In conclusion, bilirubin promotes angiogenesis through endothelial cells activation via Akt-eNOS-dependent manner.

  4. Protective Effect of Minocycline Against Ketamine-Induced Injury in Neural Stem Cell: Involvement of PI3K/Akt and Gsk-3 Beta Pathway

    PubMed Central

    Lu, Yang; Lei, Shan; Wang, Ning; Lu, Pan; Li, Weisong; Zheng, Juan; Giri, Praveen K.; Lu, Haixia; Chen, Xinlin; Zuo, Zhiyi; Liu, Yong; Zhang, Pengbo

    2016-01-01

    It has been suggested that ketamine cause injury during developing brain. Minocycline (MC) could prevent neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the protective effect of MC against ketamine-induced injury in neural stem cells (NSCs) from neonatal rat. Ketamine (100 μM/L) significantly inhibited NSC proliferation, promoted their differentiation into astrocytes and suppressed neuronal differentiation of NSCs. Moreover, the apoptotic level was increased following ketamine exposure. MC pretreatment greatly enhanced cell viability, decreased caspase-3-like activity, even reversed the differentiation changes caused by ketamine. To elucidate a possible mechanism of MC’ neuroprotective effect, we investigated the phosphatidylinositol 3-kinase (PI3K) pathway using LY294002, a specific PI3K inhibitor. Immunoblotting revealed that MC enhanced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (Gsk-3β). Our results suggest that PI3K/Akt and Gsk-3β pathway are involved in the neuroprotective effect of MC. PMID:28066173

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

    PubMed

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

    2014-05-01

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

  6. Insulin dependent apolipoprotein B degradation and phosphatidylinositide 3-kinase activation with microsomal translocation are restored in McArdle RH7777 cells following serum deprivation

    PubMed Central

    Sparks, Janet D.; Magra, Amy L.; Chamberlain, Jeffrey M.; O’Dell, Colleen; Sparks, Charles E.

    2015-01-01

    Previous studies in rat hepatocytes demonstrated that insulin-dependent apolipoprotein (apo) B degradation (IDAD) is lost when cells are maintained for 3 d under enriched culture conditions. Loss of IDAD correlates with increased expression of protein tyrosine phosphatase 1B (PTP1B) known to be associated with resistance to insulin signaling in the liver. McArdle RH7777 hepatoma (McA) cells cultured in serum containing medium are resistant to IDAD; demonstrate a 30% increase in apo B secretion, and express increased levels of PTP1B protein and mRNA. In addition, insulin-stimulated Class I phosphatidylinositide 3-kinase (PI3K) activity of anti-pY immunoprecipitates is severely blunted. IDAD resistance in McA cells correlates with diminished translocation of insulin-stimulated pY-IRS1 to intracellular membranes. Incubation of McA cells with RK682, a protein tyrosine phosphatase inhibitor, is sufficient to restore IDAD in resistant McA cells. Overall, results further support the importance of Class I PI3K activity in IDAD, and suggest that loss of this activity is sufficient to cause resistance. Although other factors are involved in downstream events including sortilin binding to apo B, autophagy, and lysosomal degradation, loss of signal generation and reduced localization of Class I PI3K to intracellular membranes plays a significant role in IDAD resistance. PMID:26616056

  7. Inhibition of p85, the non-catalytic subunit of phosphatidylinositol 3-kinase, exerts potent antitumor activity in human breast cancer cells

    PubMed Central

    Folgiero, V; Di Carlo, S E; Bon, G; Spugnini, E P; Di Benedetto, A; Germoni, S; Pia Gentileschi, M; Accardo, A; Milella, M; Morelli, G; Bossi, G; Mottolese, M; Falcioni, R

    2012-01-01

    The phosphoinositide 3-kinases (PI3Ks) are heterodimers consisting of the catalytic subunit p110 and the regulatory subunit p85. The PI3K/Akt pathway is strongly deregulated in breast cancer (BC) representing one of the mechanisms of resistance to therapies. Therefore, the identification of inhibitors of PI3K components represents one of the main goals to produce therapeutic agents. Here, we evaluated the efficacy of a phosphopeptide 1257 (P-1257) that targeting p85 strongly inhibits PI3K activity. We tested the effects of P-1257 administration in vitro and in vivo using BC cells expressing different levels of ErbB-2 and resistant or responsive to Trastuzumab. We demonstrated that inhibition of p85 activity by P-1257 induces cell death and sensitizes JIMT-1 and KPL-4 ErbB-2-overexpressing BC cells to Trastuzumab treatment. It is noteworthy that P-1257 delivery in vivo by electroporation or liposomes significantly inhibits the proliferation of tumor cells engrafted at subcutaneous and visceral sites. Overall, our data indicate that the p85 subunit is a valid target for therapeutic approaches and suggest that the structure of the peptide used in our study could be utilized for the development of novel drugs to apply in combination with therapies that fail to cure BCs with high PI3K activity. PMID:23222510

  8. Phosphatidylinositol 3-kinase and NF-kappa B/Rel are at the divergence of CD40-mediated proliferation and survival pathways.

    PubMed

    Andjelic, S; Hsia, C; Suzuki, H; Kadowaki, T; Koyasu, S; Liou, H C

    2000-10-01

    CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-kappaB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-kappaB/Rel activation. This is achieved via activation of AKT and the degradation of IkappaBalpha. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-kappaB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-kappaB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-kappaB/Rel and PI-3K are crucial for CD40-induced proliferation.

  9. Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

    PubMed Central

    Baer, Romain; Cintas, Célia; Dufresne, Marlène; Cassant-Sourdy, Stéphanie; Schönhuber, Nina; Planque, Laetitia; Lulka, Hubert; Couderc, Bettina; Bousquet, Corinne; Garmy-Susini, Barbara; Vanhaesebroeck, Bart; Pyronnet, Stéphane; Saur, Dieter; Guillermet-Guibert, Julie

    2014-01-01

    Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment. PMID:25452273

  10. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  11. PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma

    PubMed Central

    Kanteti, Rajani; Riehm, Jacob J.; Dhanasingh, Immanuel; Lennon, Frances E.; Mirzapoiazova, Tamara; Mambetsariev, Bolot; Kindler, Hedy L.; Salgia, Ravi

    2016-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent. PMID:27623107

  12. The phosphoinositide 3-kinase/Akt-signal pathway mediates proliferation and secretory function of hepatic sinusoidal endothelial cells in rats after partial hepatectomy

    SciTech Connect

    Chen Ping . E-mail: chenping@263.net; Zhang Lin; Ding Jiming; Zhu Jin; Li Ying; Duan Shigang; Yan Hongtao; Huan Yongwei; Dong Jiahong

    2006-04-14

    Objective: To investigate the role of AKT signaling pathway in hepatic sinusoidal endothelial cells (SECs) early after partial hepatectomy in rats and the regulatory mechanisms involved. Methods: The animal model of 70% hepatectomy was made. Hepatic SECs were isolated and cultured according to Braet et al.'s method with some modifications. The cultured hepatic SECs were divided into two groups: 70% partial hepatectomy groups and LY294002 group (LY). We observed the expressions of AKT and NF-{kappa}B in cultured hepatic SECs by Western blot, measured the levels of NO, NOs, IL-6, and HGF in the supernatants of hepatic SEC cultures and [{sup 3}H]thymidine incorporation, and analyzed cell cycle of cultured hepatic SECs by flow cytometer. The relationship of the Akt pathway with secretions and proliferation of hepatic SECs after partial hepatectomy was probed. Results: The levels of Akt protein expression increased significantly after partial hepatectomy in OG group and with a peak at 24 h post operation. Meanwhile, there was a markedly increase in phosphorylated Akt protein during 2-72 h after operation. But the expression and activity of Akt protein did not change significantly after partial hepatectomy in the LY group. So, partial hepatectomy can marked induce Akt expression and result in rapid and marked phosphorylation of Akt from 2 to 72 h thereafter. The changes of NF-{kappa}B expression in cultured hepatic SECs were similar to those of Akt expression after operation. The concentrations of HGF and IL-6 in the supernatants of cultured hepatic SECs were relatively low in the LY group, and were markedly increased after partial hepatectomy, with a peak at 24 h in the OG group. There were significant differences between the OG and LY groups at 6 and 24 h (P < 0.05). Both NO and NOS secretion was increased in the OG group compared to the LY group within 24 h after partial hepatectomy. But the secretion of NO and NOS was increased more markedly in the LY group than that

  13. Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

    PubMed Central

    Lee-Kwon, W; Park, D; Bernier, M

    1998-01-01

    Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase. PMID:9531502

  14. PI3 kinase is involved in cocaine behavioral sensitization and its reversal with brain area specificity

    SciTech Connect

    Zhang Xiuwu . E-mail: xwzhang@duke.edu; Mi Jing; Wetsel, William C.; Davidson, Colin; Xiong Xieying; Chen Qiang; Ellinwood, Everett H.; Lee, Tong H.

    2006-02-24

    Phosphatidylinositol 3-kinase (PI3K) is an important signaling molecule involved in cell differentiation, proliferation, survival, and phagocytosis, and may participate in various brain functions. To determine whether it is also involved in cocaine sensitization, we measured the p85{alpha}/p110 PI3K activity in the nuclear accumbens (NAc) shell, NAc core, and prefrontal cortex (PFC) following establishment of cocaine sensitization and its subsequent reversal. Naive rats were rank-ordered and split into either daily cocaine or saline pretreatment group based on their locomotor responses to an acute cocaine injection (7.5 mg/kg, i.p.). These two groups were then injected with cocaine (40 mg/kg, s.c.) or saline for 4 consecutive days followed by 9-day withdrawal. Cocaine sensitization was subsequently reversed by 5 daily injections of the D{sub 1}/D{sub 2} agonist pergolide (0.1 mg/kg, s.c.) in combination with the 5-HT{sub 3} antagonist ondansetron (0.2 mg/kg, s.c., 3.5 h after pergolide injection). After another 9-day withdrawal, behavioral cocaine sensitization and its reversal were confirmed with an acute cocaine challenge (7.5 mg/kg, i.p.), and animals were sacrificed the next day for measurement of p85{alpha}/p110 PI3K activity. Cocaine-sensitized animals exhibited increased PI3K activity in the NAc shell, and this increase was reversed by combined pergolide/ondansetron treatment, which also reversed behavioral sensitization. In the NAc core and PFC, cocaine sensitization decreased and increased the PI3K activity, respectively. These changes, in contrast to that in the NAc shell, were not normalized following the reversal of cocaine-sensitization. Interestingly, daily injections of pergolide alone in saline-pretreated animals induced PI3K changes that were similar to the cocaine sensitization-associated changes in the NAc core and PFC but not the NAc shell; furthermore, these changes in saline-pretreated animals were prevented by ondansetron given 3.5 h after

  15. Pooled Analysis of Phosphatidylinositol 3-kinase Pathway Variants and Risk of Prostate Cancer

    PubMed Central

    Koutros, Stella; Schumacher, Fredrick R.; Hayes, Richard B.; Ma, Jing; Huang, Wen-Yi; Albanes, Demetrius; Canzian, Federico; Chanock, Stephen J.; Crawford, E. David; Diver, W. Ryan; Feigelson, Heather Spencer; Giovanucci, Edward; Haiman, Christopher A.; Henderson, Brian E.; Hunter, David J.; Kaaks, Rudolf; Kolonel, Laurence N.; Kraft, Peter; Le Marchand, Loïc; Riboli, Elio; Siddiq, Afshan; Stampfer, Mier J.; Stram, Daniel O.; Thomas, Gilles; Travis, Ruth C.; Thun, Michael J.; Yeager, Meredith; Berndt, Sonja I.

    2010-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway regulates various cellular processes, inc