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Inhibitor LY294002 (inhibitor + ly294002)
Kinds of Inhibitor LY294002 Selected AbstractsPost-ischaemic activation of kinases in the pre-conditioning-like cardioprotective effect of the platelet-activating factorACTA PHYSIOLOGICA, Issue 3 2009C. Penna Abstract Aim:, Platelet-activating factor (PAF) triggers cardiac pre-conditioning against ischemia/reperfusion injury. The actual protection of ischaemic pre-conditioning occurs in the reperfusion phase. Therefore, we studied in this phase the kinases involved in PAF-induced pre-conditioning. Methods:, Langendorff-perfused rat hearts underwent 30 min of ischaemia and 2 h of reperfusion (group 1, control). Before ischaemia, group 2 hearts were perfused for 19 min with PAF (2 × 10,11 m); groups 3,5 hearts were co-infused during the initial 20 min of reperfusion, with the protein kinase C (PKC) inhibitor chelerythrine (5 × 10,6 m) or the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (5 × 10,5 m) and atractyloside (2 × 10,5 m), a mitochondrial permeability transition pore (mPTP) opener respectively. Phosphorylation of PKC,, PKB/A,t, GSK-3, and ERK1/2 at the beginning of reperfusion was also checked. Left ventricular pressure and infarct size were determined. Results:, PAF pre-treatment reduced infarct size (33 ± 4% vs. 64 ± 5% of the area at risk of control hearts) and improved pressure recovery. PAF pre-treatment enhanced the phosphorylation/activation of PKC,, PKB/A,t and the phosphorylation/inactivation of GSK-3, at reperfusion. Effects on ERK1/2 phosphorylation were not consistent. Infarct-sparing effect and post-ischaemic functional improvement induced by PAF pre-treatment were abolished by post-ischaemic infusion of either chelerythrine, LY294002 or atractyloside. Conclusions:, The cardioprotective effect exerted by PAF pre-treatment involves activation of PKC and PI3K in post-ischaemic phases and might be mediated by the prevention of mPTP opening in reperfusion via GSK-3, inactivation. [source] CSF-1 and PI 3-kinase regulate podosome distribution and assembly in macrophagesCYTOSKELETON, Issue 3 2006Ann P. Wheeler Abstract Podosomes are actin-rich adhesive foci found in several cell types, including macrophages. They have a core containing actin and actin-binding proteins and a peripheral ring of integrins and associated proteins. We show that podosomes are abundant in polarized mouse bone marrow-derived macrophages (BMM) and are found primarily in lamellae. We investigated the effects of CSF-1, which induces membrane ruffling, cell spreading, and subsequent polarization and migration, on podosome formation. CSF-1 induces a transient increase in podosome number and enhances the formation of circular arrays of podosomes. Conversely, CSF-1 withdrawal leads to a reduction in podosomes and a decrease in polarized cells. The PI 3-kinase inhibitor LY294002 induces loss of podosomes together with rapid retraction of lamellae and loss of polarity. Our results indicate that CSF-1 acts via PI 3-kinase to enhance podosome assembly and that this is linked to macrophage polarization. Cell Motil. Cytoskeleton, 2006. © 2006 Wiley-Liss, Inc. [source] Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cellsFEBS JOURNAL, Issue 8 2004In Duk Jung Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3K,, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3K, signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3K, and Rac1/Cdc42. [source] Down-regulation of the PI3-kinase/Akt pathway by ERK MAP kinase in growth factor signalingGENES TO CELLS, Issue 9 2008Hideko Hayashi The ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. However, it has not been fully understood how these two pathways interact with each other. Here, we demonstrate that inhibition of the ERK pathway by the MEK inhibitor U0126 or PD98059 significantly potentiates EGF- and FGF-induced Akt phosphorylation at both Thr308 and Ser473. We also show that hyperactivation of the ERK pathway greatly attenuates EGF- and FGF-induced Akt phosphorylation. Furthermore, the enhanced Akt phosphorylation induced by U0126 is inhibited by the PI3-kinase inhibitor LY294002, and is accompanied by the up-regulation of Ras activity. These results suggest that the ERK pathway inhibition enhances Akt phosphorylation through the Ras/PI3-kinase pathway. Thus, our results demonstrate that the ERK pathway negatively modulates the PI3-kinase/Akt pathway in response to growth factor stimulation. [source] The phosphatidylinositol-3 kinase (PI3K)-Akt pathway suppresses neurite branch formation in NGF-treated PC12 cellsGENES TO CELLS, Issue 8 2003Maiko Higuchi Background:, Previous studies have shown that phosphatidylinositol-3 kinase (PI3K) plays an important role in NGF (nerve growth factor)-induced neurite elongation. However, the roles of the PI3K pathway in neurite branch formation were not fully understood. Also, it was not clear where the PI3K pathway is activated during branch formation. Results:, We found that the treatment of PC12 cells with the PI3K inhibitor LY294002 resulted in a marked increase in the number of neurite branch points, suggesting a suppressive role of PI3K in neurite branch formation. Expression of a constitutively active form of Akt, a downstream effector of PI3K, decreased the number of branch points, whereas that of a dominant-negative form of Akt increased it. In contrast, inhibition of neither Rac, mTOR nor GSK3, other effectors of PI3K, promoted branch formation. Importantly, the phosphorylated form of endogenous Akt was localized at the tips of growth cones, but devoid of small branches in NGF-treated PC12 cells. A GFP-fusion protein of the plekstrin-homology (PH) domain of Akt was also localized at the tips of growth cones. Conclusions:, The PI3K-Akt pathway thus plays a key role in suppression of neurite branch formation in NGF-treated PC12 cells. Summary figure, Figure Summary figure,. working model for the regulation of neuritogenesis in PC12 cells. PI3K may mediate NGF regulation of neuritogenesis via two pathways. Rac induces neurite elongation and branch formation. Akt induces neurite elongation, but prevents branch formation. [source] The TLR3 ligand polyI:C downregulates connexin 43 expression and function in astrocytes by a mechanism involving the NF-,B and PI3 kinase pathwaysGLIA, Issue 8 2006Yongmei Zhao Abstract Toll-like receptor 3 (TLR3) is a component of the innate immune response that responds to dsRNA viruses and virus replication intermediates. In this study we show that activation of astrocytes with the dsRNA mimetic polyinosinic-cytidylic acid (pI:C) results in loss of expression of connexin43 (Cx43) mRNA and protein while upregulating the expression of the ionotropic P2 receptor P2X4R. Analysis of the signaling pathways involved failed to demonstrate a role for the p38 MAP kinase, ERK, or JNK signaling pathways whereas an inhibitor of the PI3 kinase/Akt pathway effectively blocked the action of pI:C. Using adenoviral vectors containing a super-repressor of NF-,B (NF-,B SR) construct or a dominant negative interferon regulatory factor 3 (dnIRF3) construct showed that inhibition of both transcription factors also blocked the effects of pI:C. To explore the functional consequences of pI:C activation we used a pore-forming assay for P2X4R activity and a scrape loading assay for gap junction intercellular communication (GJIC). No pore-forming activity consistent with functional P2X4R expression was detected in either control or activated astrocytes. In contrast, robust Lucifer yellow transfer indicative of GJIC was detected in resting cells that was lost following pI:C activation. The dnIRF3 construct failed to restore GJIC whereas the NF-,B SR or the NF-,B inhibitor BAY11-7082 and the PI3K inhibitor LY294002 all significantly reversed the effect of pI:C on GJ connectivity. We conclude that activation of the innate immune response in astrocytes is associated with functional loss of GJIC through a pathway involving NF-,B and PI3 kinase. © 2006 Wiley-Liss, Inc. [source] Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cellsINTERNATIONAL ENDODONTIC JOURNAL, Issue 12 2005F.-M. Huang Abstract Aim, To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. Methodology, The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. Results, The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Conclusions,Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA. [source] Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2006Maarten L. Janmaat Abstract In this study, we have characterized a panel of NSCLC cell lines with differential sensitivity to gefitinib for activating mutations in egfr, pik3ca, and k-ras, and basal protein expression levels of PTEN. The egfr mutant NSCLC cell line H1650 as well as the egfr wild type cell lines H292 and A431 were highly sensitive to gefitinib treatment, indicating that other factors determine gefitinib-sensitivity in egfr wild type cells. Activating k-ras mutations were specifically detected in gefitinib-resistant cells, suggesting that the occurrence of k-ras mutations is correlated with resistance to EGFR antagonists. No pik3ca mutations were detected within the panel of cell lines, and PTEN protein expression levels did not correlate with gefitinib sensitivity. Gefitinib effectively blocked Akt and Erk phosphorylation in two gefitinib-sensitive NSCLC cell lines, further supporting our previous findings that persistent activity of the PI3K/Akt and/or Ras/Erk pathways is associated with gefitinib-resistance of NSCLC cell lines. Gefitinib-resistant NSCLC cell lines, showing EGFR-independent activity of the PI3K/Akt or Ras/Erk pathways, were treated with gefitinib in combination with specific inhibitors of mTOR, P13K, Ras, and MEK. Additive cytotoxicity was observed in A549 cells co-treated with gefitinib and the MEK inhibitor U0126 or the farnesyl transferase inhibitor SCH66336 and in H460 cells treated with gefitinib and the PI3K inhibitor LY294002, but not in H460 cells treated with gefitinib and rapamycin. These data suggest that combination treatment of NSCLC cells with gefitinib and specific inhibitors of the PI3K/Akt and Ras/Erk pathways may provide a successful strategy. © 2005 Wiley-Liss, Inc. [source] Nerve growth factor blocks thapsigargin-induced apoptosis at the level of the mitochondrion viaregulation of BimJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6a 2008E. Szegezdi Abstract This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+ -ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (,3, ,7 and ,9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bimel mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. [source] Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1, (HIF-1,) through inhibiting protein synthesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Dae-Hee Lee Abstract Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1, (HIF-1,) in normoxia. In this study, under hypoxic conditions (1% O2), we examined the effect of quercetin on the intracellular level of HIF-1, and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1, accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1, accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O2) in the presence of 100 µM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1, accumulation were observed under hypoxic conditions. Treatment with 100 µM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1, accumulation during hypoxia. These results suggest that suppression of HIF-1, accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. J. Cell. Biochem. 105: 546,553, 2008. © 2008 Wiley-Liss, Inc. [source] Interleukin-1, enhances nucleotide-induced and ,-secretase-dependent amyloid precursor protein processing in rat primary cortical neurons via up-regulation of the P2Y2 receptorJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Qiongman Kong Abstract The heterologous expression and activation of the human P2Y2 nucleotide receptor (P2Y2R) in human 1321N1 astrocytoma cells stimulates ,-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPP,). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y2R-mediated production of sAPP, occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1, (IL-1,) up-regulated both P2Y2R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y2R was abrogated by pre-incubation with Bay 11-7085, an I,B-, phosphorylation inhibitor, which suggests that P2Y2R mRNA transcript levels are regulated through nuclear factor-,-B (NF,B) signaling. Furthermore, the P2Y2R agonist Uridine-5,-triphosphate (UTP) enhanced the release of sAPP, in rPCNs treated with IL-1, or transfected with P2Y2R cDNA. UTP-induced release of sAPP, from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y2R-mediated release of sAPP, from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1,, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y2R in neurons. [source] Activation of phosphoinositide-3 kinase/Akt pathway by FeSO4 in rat cerebral cortex synaptic endingsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Romina M. Uranga Abstract The aim of this work was to study the involvement of the phosphoinositide-3-kinase (PI3K)/Akt pathway in synaptic endings incubated under oxidative stress conditions. Synaptosomes purified from rat cerebral cortex were exposed to FeSO4 (50 ,M) for different periods of time. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenate (LDH) leakage were significantly affected after 5 min of incubation in the presence of FeSO4, with respect to control conditions. In whole synaptosomes incubated in the presence of [,- 32P]ATP, phosphoinositide (PPI) labeling was increased after 5 min of Fe2+ exposure. This effect was prevented by the specific PI3K inhibitor LY294002. Anti-p85 immunoprecipitates (IPs) obtained from synaptosomes preincubated with Fe2+ (5 min) showed a PI3K activity two-fold higher than the activity recovered under control conditions. Additionally, Akt activation was temporally coincident with PI3K activation. LY294002 was not able to prevent the LDH leakage and diminution of MTT reduction induced by Fe2+. Our results demonstrate that free iron provokes the early activation of PI3K/Akt pathway, but this activation is not sufficient for protecting synaptic endings from oxidative damage. © 2007 Wiley-Liss, Inc. [source] Signal transduction pathways involved in the stimulation of tissue type plasminogen activator by interleukin-1, and Porphyromonas gingivalis in human osteosarcoma cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2006Yu-Chao Chang Background:, Recently, evidences have shown that tissue type plasminogen activator (t-PA) may play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human osteosarcoma cells are not fully understood. Objectives:, The purpose of this study was to investigate the caseinolytic activity in human osteosarcoma cell line U2OS cells stimulated with interleukin-1, (IL-1,) or Porphyromonas gingivalis in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Methods:, IL-1, and the supernatants of P. gingivalis were used to evaluate the caseinolytic activity in U2OS cells by using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the caseinolytic activity. Results:, Casein zymography exhibited a caseinolytic band with a molecular weight of approximately 70 kDa, suggestive of the presence of t-PA. Secretion of t-PA was found to be stimulated with IL-1, and P. gingivalis during a 2-day culture period (p < 0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1, or P. gingivalis -stimulated t-PA production, respectively (p < 0.05). Conclusions:, Our findings demonstrated that IL-1, and P. gingivalis enhance t-PA production in human osteosarcoma cells, and that the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of t-PA. SB203580, U0126, and LY294002 suppress t-PA production and/or activity and may therefore be valuable therapeutics in t-PA-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis. [source] Participation of various kinases in staurosporine-induced apoptosis of RAW 264.7 cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2002Kouya Yamaki Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage-like cell line, as determined by DNA fragmentation, the increase of annexin V-stained cells, and the cleavage of poly(ADP- ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub-G1 cells revealed that the DNA fragmentation occurred in a time- and concentration-dependent manner at 0.021,2.1 ,m of staurosporine. Staurosporine induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal-regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002 potentiated the staurosporine-induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H-89 potentiated the staurosporine-induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro-31,8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine-induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and P13K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation. [source] Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathwayMOLECULAR CARCINOGENESIS, Issue 11 2007Xingya Wang Abstract Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1,4). In this study, we investigated the role of EP receptors in PGE2 -induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM,10 µM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2 -induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2,5,-dideoxyadenosine, at concentrations that inhibited PGE2 -induced cAMP, significantly blocked PGE2 -induced VEGF secretion in PC-3 cells. We conclude that PGE2 -induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. © 2007 Wiley-Liss, Inc. [source] TG101209, a novel JAK2 inhibitor, has significant in vitro activity in multiple myeloma and displays preferential cytotoxicity for CD45+ myeloma cells,AMERICAN JOURNAL OF HEMATOLOGY, Issue 9 2010Vijay Ramakrishnan Interaction of myeloma cells with the bone marrow microenvironment is mediated in large part through different cytokines, especially VEGF and IL6. These cytokines, especially IL6, leads to upregulation of the JAK/STAT pathway in myeloma cell, contributing to increased proliferation, decreased apoptosis, and acquired drug resistance. Here, we examined the preclinical activity of a novel JAK2 inhibitor TG101209. TG101209 induced dose- and time-dependent cytotoxicity in a variety of multiple myeloma (MM) cell lines. The induction of cytotoxicity was associated with inhibition of cell cycle progression and induction of apoptosis in myeloma cell lines and patient-derived plasma cells. Evaluation of U266 cell lines and patient cells, which have a mix of CD45 positive and negative cells, demonstrated more profound cytotoxicity and antiproliferative activity of the drug on the CD45+ population relative to the CD45, cells. Exploring the mechanism of action of TG101209 indicated downregulation of pJak2, pStat3, and Bcl-xl levels with upregulation of pErk and pAkt levels indicating cross talk between signaling pathways. TG101209, when used in combination with the PI3K inhibitor LY294002, demonstrated synergistic cytotoxicity against myeloma cells. Our results provide the rationale for clinical evaluation of TG101209 alone or in combination with PI3K/Akt inhibitors in MM. Am. J. Hematol., 2010. © 2010 Wiley-Liss, Inc. [source] Restoration of PTEN expression alters the sensitivity of prostate cancer cells to EGFR inhibitors,THE PROSTATE, Issue 9 2008Z. Wu Abstract Introduction Prostate cancer (CaP) progression from an androgen-dependent to an androgen-independent state is associated with overexpression of EGFR family members or activation of their downstream signaling pathways, such as PI3K-Akt and MAPK. Although there are data implicating PI3K-Akt or MAPK pathway activation with resistance to EGFR inhibitors in CaP, the potential cross-talk between these pathways in response to EGFR or MAPK inhibitors remains to be examined. Methods Cross-talk between PTEN and MAPK signaling and its effects on CaP cell sensitivity to EGFR or MAPK inhibitors were examined in a PTEN-null C4-2 CaP cell, pTetOn PTEN C4-2, where PTEN expression was restored conditionally. Results Expression of PTEN in C4-2 cells exposed to EGF or serum was associated with increased phospho-ERK levels compared to cells without PTEN expression. Similar hypersensitivity of MAPK signaling was observed when cells were treated with a PI3K inhibitor LY294002. This enhanced sensitivity of MAPK signaling in PTEN-expressing cells was associated with a growth stimulatory effect in response to EGF. Furthermore, EGFR inhibitors gefitinib and lapatinib abrogated hypersensitivity of MAPK signaling and cooperated with PTEN expression to inhibit cell growth in both monolayer and anchorage-independent conditions. Similar cooperative growth inhibition was observed when cells were treated with the MEK inhibitor, CI1040, in combination with PTEN expression suggesting that inhibition of MAPK signaling could mediate the cooperation of EGFR inhibitors with PTEN expression. Conclusions Our results suggest that signaling cross-talk between the PI3K-Akt and MAPK pathways occurs in CaP cells, highlighting the potential benefit of targeting both the PI3K-Akt and MAPK pathways in CaP treatment. Prostate 68:935,944, 2008. © 2008 Wiley-Liss, Inc. [source] Extracellular signal-regulated kinase (ERK) activation in chicken heterophils stimulated with phorbol 12-myristate 13-acetate (PMA), Formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS)ANIMAL SCIENCE JOURNAL, Issue 5 2009GERILECHAOGETU ABSTRACT The signaling pathways leading to the activation of extracellular signal-regulated kinase (ERK) by phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) in chicken heterophils were examined. To determine the mechanism of ERK's activation and its relation with the influx of calcium ions, heterophils were stimulated by PMA, fMLP and LPS. ERK was not activated by fMLP. LPS- and PMA-stimulated activation of ERK, based on Western blotting with antibodies against the phosphorylated form of ERK, was attenuated by the pretreatment of cells with the intracellular calcium chelator BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N,,N,-tetraacetic acid) but not with the extracellular calcium chelator EGTA (glycol-bis(2-aminoethylether)-N,N,N,,N,-tetraacetic acid). Exposure of cells to the protein kinase C (PKC) inhibitor GF109203X inhibited the LPS- and PMA-stimulated phosphorylation of ERK in a concentration-dependent manner. The LPS-stimulated phosphorylation was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results indicate that the LPS-induced phosphorylation of ERK in the chicken heterophils is mediated by PLC, PKC and intracellular calcium, and the PMA-stimulated phosphorylation is dependent on intracellular calcium ion and PKC. [source] Purple Sweet Potato Color Alleviates D-galactose-induced Brain Aging in Old Mice by Promoting Survival of Neurons via PI3K Pathway and Inhibiting Cytochrome C-mediated ApoptosisBRAIN PATHOLOGY, Issue 3 2010Jun Lu Abstract Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, protects brain function against oxidative stress induced by D-galactose (D-gal) (Sigma-Aldrich, St. Louis, MO, USA). Our data showed that PSPC enhanced open-field activity, decreased step-through latency, and improved spatial learning and memory ability in D-gal-treated old mice by decreasing advanced glycation end-products' (AGEs) formation and the AGE receptor (RAGE) expression, and by elevating Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (Sigma-Aldrich) and catalase (CAT) expression and activity. Cleavage of caspase-3 and increased terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in D-gal-treated old mice were inhibited by PSPC, which might be attributed to its antioxidant property. PSPC also suppressed the activation of c-Jun NH2 -terminal kinase (JNK) and the release of cytochrome c from mitochondria that counteracted the onset of neuronal apoptosis in D-gal-treated old mice. Furthermore, it was demonstrated that phosphoinositide 3-kinase (PI3K) activation was required for PSPC to promote the neuronal survival accompanied with phosphorylation and activation of Akt and p44/42 mitogen-activated protein kinase (MAPK) by using PI3K inhibitor LY294002 (Cell Signaling Technology, Inc., Beverly, MA, USA), implicating a neuronal survival mechanism. The present results suggest that neuronal survival promoted by PSPC may be a potentially effective method to enhance resistance of neurons to age-related disease. [source] R-Ras promotes tumor growth of cervical epithelial cellsCANCER, Issue 3 2003Héctor Rincón-Arano B.S. Abstract BACKGROUND R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of ,1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K. Cancer 2003;97:575,85. © 2003 American Cancer Society. DOI 10.1002/cncr.11093 [source] CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytesCELL PROLIFERATION, Issue 4 2007L. Wierød In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G1 phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. Methods and results: In this study, we have explored the role of CDK4 activity during G1 progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. Conclusions: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G1 phase. [source] Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitroCELLULAR MICROBIOLOGY, Issue 2 2009Tobias Tenenbaum Summary Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an ,inverted' Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerobrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, but not by its inactive analogue Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3-kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood,cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system. [source] Thyroid hormone-mediated growth and differentiation of growth plate chondrocytes involves IGF-1 modulation of ,-catenin signalingJOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2010Lai Wang Abstract Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/,-catenin signaling pathway. Insulin-like growth factor 1 (IGF-1) has been described as a stabilizer of ,-catenin, and thyroid hormone is a known stimulator of IGF-1 receptor expression. The purpose of this study was to test the hypothesis that IGF-1 signaling is involved in the interaction between the thyroid hormone and the Wnt/,-catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF-1 and the IGF- receptor (IGF1R) stimulate Wnt-4 expression and ,-catenin activation in growth plate chondrocytes. The positive effects of IGF-1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T3 activates IGF-1/IGF1R signaling and IGF-1-dependent PI3K/Akt/GSK-3, signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T3 -mediated Wnt-4 expression, ,-catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and ,-catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF-1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF-1/IGF1R-mediated PI3K/Akt/GSK3, pathway, cell hypertrophy is likely due to activation of Wnt/,-catenin signaling, which is at least in part initiated by IGF-1 signaling or the IGF-1-activated PI3K/Akt signaling pathway. © 2010 American Society for Bone and Mineral Research [source] Opposing Actions of Phosphatidylinositol 3-Kinase and Glycogen Synthase Kinase-3, in the Regulation of HSF-1 ActivityJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Gautam N. Bijur Abstract: Elevated temperatures activate the survival promoters Aktand heat shock factor-1 (HSF-1), a transcription factor that induces theexpression of heat shock proteins (HSPs), such as HSP-70. Because neuronalmechanisms controlling these responses are not known, these were investigatedin human neuroblastoma SH-SY5Y cells. Heat shock (45°C) rapidly activatedAkt, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, butonly Akt was activated in a phosphatidylinositol 3-kinase (PI-3K)-dependentmanner, as the PI-3K inhibitors LY294002 and wortmannin blocked Aktactivation, but not ERK1/2 or p38 activation. Akt activation was not blockedby inhibition of p38 or ERK1/2, indicating the independence of these signalingsystems. Heat shock treatment also caused a rapid increase in HSF-1 DNAbinding activity that was partially dependent on PI-3K activity, as both thePI-3K inhibitors attenuated this response. Because Akt inhibits glycogensynthase kinase-3, (GSK-3,), an enzyme that facilitates cell death,we tested if GSK-3, is a negative regulator of HSF-1 activation.Overexpression of GSK-3, impaired heat shock-induced activation of HSF-1,and also reduced HSP-70 production, which was partially restored by theGSK-3, inhibitor lithium. Thus, heat shock-induced activation of PI-3Kand the inhibitory effect of GSK-3, on HSF-1 activation and HSP-70expression imply that Akt-induced inhibition of GSK-3, contributes to theactivation of HSF-1. [source] | |||||||||||||||||||||||||