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PubMed2006

Signalling molecules essential for neuronal survival and differentiation.

Motoneurons are made in excess throughout development. Initial analysis of the mechanisms that lead to apoptotic cell death during later stages of development and the early postnatal period led to the discovery of neurotrophic factors. These factors comprise different families acting through different tyrosine kinase receptors. Intracellular signalling cascades that lead to the survival of neurons are, on the one hand, the Ras/Raf (Ras-activated factor)/MAPK (mitogen-activated protein kinase) pathway and, on the other, the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) pathway. The initial thought of these factors acting as single molecules in separate cascades has been converted into a model in which the dynamics of interaction of these pathways and the subcellular diverse functions of the key regulators have been taken into account. Bag1 (Bcl-2-associated athanogene 1), a molecule that was originally found to act as a co-chaperone of Hsp70 (heat-shock protein 70), also interacts with B-Raf, C-Raf and Akt to phosphorylate Bad (Bcl-2/Bcl-X(L)-antagonist, causing cell death), a pro-apoptotic member of the Bcl-2 family, and leads to specific subcellular distribution of phosphorylated Akt and B-Raf. These functions lead to survival of embryonic neural stem cells and therefore serve as a key event to regulate the viability of these cells.

Animals↗
PubMed2002

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae.

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.

Apoptosis↗
PubMed2026

Carrot Juice Intake Modulates Oncogenic and Inflammatory Pathways in Advanced Colorectal Adenomas: A Pilot Feasibility Study.

Carrots are a rich dietary source of carotenoids and polyacetylenes, bioactive compounds with demonstrated anti-inflammatory and anticancer properties in experimental models. Epidemiological evidence suggests that carrot consumption is associated with a reduced risk of colorectal cancer; however, clinical data linking carrot intake to molecular changes in premalignant colorectal tissue remain limited. In this pilot intervention study, 20 patients with advanced colorectal adenomas were enrolled. Fifteen participants consumed carrot juice daily for 21 days, while five served as untreated controls. Paired adenoma biopsies were collected before and after the intervention and were analyzed using gene expression microarrays to assess transcriptional responses. Carrot juice intake was well tolerated, with adherence exceeding 95% and no reported adverse events. Transcriptomic analysis revealed modulation of key pathways implicated in colorectal carcinogenesis, including downregulation of the WNT, PI3K-AKT, and MAPK signaling pathways, as well as cyclooxygenase-2-related inflammatory pathways and cytokine signaling. These changes were consistent with reduced oncogenic signaling and attenuation of inflammatory activity within adenoma tissue. In summary, short-term carrot juice consumption was associated with coordinated suppression of molecular pathways involved in colorectal adenoma progression. These findings provide preliminary clinical evidence that a whole-food dietary intervention may influence early carcinogenic processes and support the need for larger controlled studies evaluating clinical outcomes.

Journal Article↗
PubMed2002

Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent G(s) signaling during beta2-adrenergic stimulation.

Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of beta2-adrenoceptor (beta2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of beta2-AR-coupled G(i) proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the beta2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the beta2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables beta2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in beta2-AR-induced cAMP formation. Blocking G(i) or Gbetagamma signaling with pertussis toxin or betaARK-ct, a peptide inhibitor of Gbetagamma, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of beta2-AR-PKA signaling sequentially involves G(i), Gbetagamma, and PI3K. Thus, PI3K constitutes a key downstream event of beta2-AR-G(i) signaling, which confines and negates the concurrent beta2-AR/G(s)-mediated PKA signaling.

Adrenergic beta-Agonists↗
PubMed2003

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways.

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.

Animals↗
PubMed2025

Trichomonas vaginalis extracellular vesicles activate the NLRP3 inflammasome and TLR3-mediated inflammatory cascades in host cells.

Trichomonas vaginalis (TV) is a flagellated parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection (STI), with over 275 million cases annually. TV has been shown to secrete extracellular vesicles (TV-EVs) to regulate intercellular communication between parasites and host immune response; however, the mechanisms by innate immunity against TV-EVs are largely unknown. Herein, we aim to investigate the molecular mechanisms of inflammation induced by TV-EVs and identify novel proteins modulating the immune response in host cells. Firstly, the morphological characteristics of TV-EVs have been analyzed by transmission electron microscope (TEM) and nanoparticle tracking analysis, revealing that the vesicles are round-shaped bilayer membrane structures with size mostly about 100-120 nm. Additionally, the internalization of TV-EVs by host cells has been validated through immunofluorescence and TEM analysis. The multiplex immunoassay identified that TV-EVs induce the secretion of inflammatory cytokines, including CXCL1, IL-6, IL-8 and MIP-1β in THP-1 macrophages and ectocervical cells (Ect). Mechanistically, TV-EVs induce TLR3 overexpression to activate the NF-κB/NLRP3 pathway in THP-1 macrophages. Additionally, TV-EVs activate the PI3K-mediated NF-κB, p38 MAPK and ERK pathways in Ect. Moreover, TV-EV-induced TLR3 overexpression positively regulates the PI3K and NF-κB pathways, while simultaneously suppressing the p38 MAPK and ERK pathways in Ect. Proteomic analysis identified that TV-EVs upregulate MICB and TRAF3IP2, which are also positively regulated by TLR3 and involved in TV-EV-induced inflammatory cascade. Altogether, this study significantly advances our understanding of the immunomodulatory roles of TV-EVs in host cells, paving the way for future treatment of trichomoniasis and TV-associated STIs.

Humans↗
PubMed2006

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates CD151-induced endothelial cell proliferation and cell migration.

We previously reported that CD151 promotes neovascularization and improves blood perfusion in rat hind-limb ischemia model, but the precise mechanism is still unclear. Endothelial cell proliferation and cell migration play critical roles in angiogenesis. Many growth factors and hormones have been shown to regulate cell proliferation, cell migration and angiogenesis, including the activation of eNOS activity, via the PI3K/Akt signaling pathway. Whether CD151 induces cell proliferation and cell migration via PI3K/Akt signaling pathway is not known. Here we showed that CD151 promotes human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation in vitro, accompanied by increased phosphorylation of Akt and eNOS, leading to increased eNOS activity and nitric oxide (NO) levels after rAAV-CD151 infection, whereas infection with rAAV-anti-CD151 attenuated the effects of CD151, which suggested that CD151 can activate PI3K/Akt pathway. Moreover, inhibitors of PI3K (LY294002) and eNOS (l-NAME) can attenuate CD151-induced cell proliferation and cell migration. The results suggested that activation of PI3K/Akt signaling pathway mediates CD151-induced cell proliferation and migration.

Antigens, CD↗
PubMed2006

Impairment of cardioprotective PI3K-Akt signaling by post-infarct ventricular remodeling is compensated by an ERK-mediated pathway.

Recently we found that post-infarct remodeling disrupts PI3KAkt signaling triggered by erythropoietin (EPO) but an unknown compensatory mechanism preserves EPO-induced protection against infarction in those hearts. In this study, we examined the possibility that ERK-mediated signaling is the compensatory mechanism affording protection in post-infarct remodeled hearts. Four weeks after coronary ligation in situ (post-MI group, post-MI) or a sham operation (sham group, Sham), hearts were isolated, perfused and subjected to 25-min global ischemia/2-h reperfusion. Infarct size was expressed as a percentage of risk area size (%I/R), from which scarred infarct by coronary ligation was excluded. EPO infusion (5 U/ml) before ischemia reduced %I/R similarly in Sham and post-MI (from 62.0 +/- 5.1 to 39.4 +/- 4.8 in Sham and from 58.6 +/- 6.6 to 36.3 +/- 3.8 in post-MI). PD98059, a MEK1/2 inhibitor, abolished this EPO-induced protection in post-MI (%I/R = 60.7 +/- 4.9) but not in Sham (%I/R = 35.1 +/- 5.4). EPO induced PI3K-dependent phosphorylation of Akt in Sham but not in post-MI. EPO increased phosphorylation levels of ERK1/2 both in Sham and post-MI, but this phosphorylation was diminished by a PI3K inhibitor in Sham but not in post-MI. These results suggest that PI3K-independent activation of ERK compensates the lack of signal input from the PI3K-Akt pathway to achieve EPO-induced protection in the remodeled myocardium.

Animals↗
PubMed2000

Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk.

Pancreastatin (PST), a chromogranin A-derived peptide, has counterregulatory effects on insulin in the hepatocyte and the adipocyte, suggesting a possible role in insulin resistance. The mechanism of PST action on glucose and lipid metabolism is typical of a calcium-mobilizing hormone and involves a receptor Gq/11 protein-phospholipase C (PLC)-beta pathway. In the rat adipocyte, PST inhibits insulin-mediated glucose transport, glucose utilization, and lipid synthesis, and it has a lipolytic effect but stimulates basal and insulin-stimulated protein synthesis. We have also recently studied the PST receptor-effector system in adipocyte membranes. To further investigate the mechanisms of PST effect on insulin action, we studied the cross-talk of PST with insulin signaling in the rat adipocyte. We found that PST inhibits insulin-stimulated GLUT4 translocation to the membrane, which may explain the reported inhibition of glucose transport. Tyrosine phosphorylation of the activated insulin receptor, insulin receptor substrate (IRS)-1, and p60-70 was also blunted, preventing their association with p85 phosphatidylinositol 3-kinase (PI3K) and their activity. The mechanism of this inhibition involves the activation of the "classical" protein kinase C isoforms and the serine phosphorylation of insulin receptor and IRS-1. On the other hand, PST activates the mitogen-activated protein kinase (MAPK) signaling module and enhances the effect of insulin. This pathway may account for the described effect of PST on protein synthesis. In conclusion, PST seems to inhibit the insulin-stimulated PI3K pathway in the adipocyte, whereas it activates the MAPK pathway. These data provide some clues to the PST cross-talk with insulin signaling that may explain the PST effects on glucose metabolism and protein synthesis.

Adipocytes↗
PubMed2002

Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway.

In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.

3T3 Cells↗
PubMed2005

Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast.

The mammalian signalling pathway involving class I PI3K (phosphoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase) and PKB (protein kinase B)/c-Akt has roles in multiple processes, including cell proliferation and apoptosis. To facilitate novel approaches for genetic, molecular and pharmacological analyses of these proteins, we have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote, Saccharomyces cerevisiae (yeast). High-level expression of the p110 catalytic subunit of mammalian PI3K dramatically inhibits yeast cell growth. This effect depends on PI3K kinase activity and is reversed partially by a PI3K inhibitor (LY294002) and reversed fully by co-expression of catalytically active PTEN (but not its purported yeast orthologue, Tep1). Growth arrest by PI3K correlates with loss of PIP2 (phosphatidylinositol 4,5-bisphosphate) and its conversion into PIP3 (phosphatidylinositol 3,4,5-trisphosphate). PIP2 depletion causes severe rearrangements of actin and septin architecture, defects in secretion and endocytosis, and activation of the mitogen-activated protein kinase, Slt2. In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Phospho-specific antibodies show that both active and kinase-dead PKB/c-Akt are phosphorylated at Thr308 and Ser473. Thr308 phosphorylation, but not Ser473 phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), did not block Ser473 phosphorylation, implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

TGFbeta trophic factors differentially modulate motor axon outgrowth and protection from excitotoxicity.

Transforming growth factor (TGF) beta-like trophic factors have been shown to be protective in acute neuronal injury paradigms. In the current study, we analyzed and compared members of this growing family, including glial cell line-derived neurotrophic factor (GDNF), neurturin, nodal, persephin, and TGFbeta1, for protection against chronic glutamate toxicity. In parallel, we developed a organotypic spinal cord culture system to study the ability of these factors to promote motor axon outgrowth across white matter. Using these systems, we were able to differentiate the neuroprotective effect of the TGFbeta-like factors from their motor axon outgrowth-promoting activity. GDNF, neurturin, persephin, nodal, and TGFbeta1 all protected against excitotoxic motor neuron degeneration. Low amounts of GDNF (1 ng/ml) and high concentrations of neurturin induced vigorous motor axon outgrowth. In contrast, nodal, persephin, and TGFbeta1 did not induce motor axon outgrowth. Both GDNF and neurturin bind to Ret receptor complexes and were capable of activating the MAP kinase pathway. A specific inhibitor of MAP kinase kinase, PD98059, inhibited the motor axon outgrowth-promoting activity of the GDNF but not the neuroprotective activity. Similarly, the specific PI3K inhibitors, LY294002 and wortmannin, were able to inhibit the promotion of motor axon outgrowth by GDNF, but did not affect neuroprotective activity. Our results suggest that the neurite outgrowth-promoting effect of GDNF is mediated through the PI3K and MAP kinase pathways. The neuroprotective effect of GDNF appears to be through a separate pathway.

Androstadienes↗
PubMed2001

Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation.

We investigated the role of Ras in vascular endothelial growth factor (VEGF)-mediated signal transduction and the promotion of angiogenic changes primary endothelial cells. We find that VEGF potently induces Ras activation and that this step is essential for the stimulation by VEGF of several cellular changes associated with angiogenesis, including proliferation, migration, and branching morphogenesis in three-dimensional culture. Inhibition of Ras signaling induced subtle changes in the actin architecture but had no effect on the phosphatidylinositol 3-kinase (PI3K) or p38 signaling pathways. In contrast, activation of ERK was largely dependent on Ras. Although inhibiting ERK activity completely suppressed cell proliferation and partially blocked in vitro differentiation, neither ERK nor PI3K activity was required for VEGF-induced migration. These data provide the first direct demonstration that inhibition of Ras signal transduction is anti-angiogenic. Interestingly, VEGF signal transduction bifurcates both upstream and downstream of Ras, with different Ras-dependent signals controlling endothelial cell proliferation and migration, essential components of the angiogenic response.

Adenoviridae↗
PubMed2005

Systematic evaluation of anti-apoptotic growth factor signaling in vascular smooth muscle cells. Only phosphatidylinositol 3'-kinase is important.

Peptide growth factors contribute to the pathogenesis of cardiovascular diseases by inducing a variety of cellular responses including anti-apoptotic effects. Several of the signaling molecules that are activated by growth factor receptors such as Src family kinases (Src), phosphatidylinositol 3'-kinase (PI3K), phospholipase Cgamma (PLCgamma), Ras, and SHP-2 were shown to mediate survival signals. We systematically investigated the relative contribution of each signaling molecule for growth factor-dependent cell survival in vascular smooth muscle cells (VSMC). Our approach was the use of mutated plateletderived growth factor (PDGF) beta-receptors (betaPDGFR) in which the tyrosine residues required for binding of each signaling molecule were individually mutated to phenylalanine. To bypass endogenous PDGFR in VSMC we used chimeric receptors (ChiRs), containing the extracellular domain of the macrophage colony-stimulating factor (M-CSF) receptor and the cytoplasmic domain of the wild type (WT) or mutated betaPDGFR. Selective activation of the ChiR-WT with M-CSF significantly reduced apoptosis to the same extent as PDGF-BB in non-transfected cells. Deletion of the binding site for PI3K, but not for Src, RasGAP, SHP-2, or PLCgamma, completely abolished the anti-apoptotic effect. Consistently, a ChiR mutant that only binds PI3K was fully able to mediate cell survival as efficiently as the ChiR-WT. Furthermore, the PDGF-dependent anti-apoptotic effect in non-transfected cells was completely abolished by the PI3K inhibitor wortmannin, whereas inhibitors of Src, PLCgamma, ERK, or p38 MAP kinase had no effect. The exploration of downstream signaling events revealed that PDGF-BB activates the anti-apoptotic Akt signaling pathway in a PI3K-dependent manner. Moreover, Akt phosphorylates and thus inactivates the pro-apoptotic proteins BAD and Forkhead transcription factors (FKHR, FKHRL1). We conclude that growth factor-dependent cell survival in VSMC is mediated only by activation of the PI3K/Akt pathway, whereas all other receptor-associated signaling molecules do not play a significant role.

Animals↗
PubMed2003

Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent Pathways cooperate to maintain lung cancer cell survival.

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.

Apoptosis↗
PubMed2001

Signal transduction pathways for activation of extracellular signal-regulated kinase by arachidonic acid in rat neutrophils.

Phosphorylation of extracellular signal-regulated kinase (ERK) in response to arachidonic acid (AA) was rapid and transient, peaking at 1 min and disappearing after 3 min, and it was accompanied by an increase in ERK activity in rat neutrophils. We examined the upstream regulation of AA-stimulated ERK activation using one of the following signaling pathway inhibitors to pretreat rat cells: the ERK kinase inhibitor U0126 or PD98059, the G(i/o) inhibitor pertussis toxin (PTX), the tyrosine kinase inhibitor genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or LY294002, the Ca2+ chelator 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, or the phospholipase C (PLC) inhibitor U73122. All of these inhibitors attenuated AA-induced ERK activation. Activation of ERK was also effectively attenuated by the cyclooxygenase and lipoxygenase inhibitor BW755C and by the leukotriene biosynthesis inhibitor MK886, but the cyclooxygenase inhibitor indomethacin did not attenuate ERK activation. After exposing cells to three distinct protein kinase C (PKC) inhibitors, we found that Gö6976 significantly attenuated ERK phosphorylation but potentiated ERK activity. Neither Gö6983 nor GF109203X affected AA-induced responses. These data suggest that the lipoxygenase metabolite(s) produced mediates AA-stimulated ERK activation and that this effect is upstream regulated by PT-sensitive G protein, non-receptor tyrosine kinase, PI3K, and PLC/Ca2+ signaling pathways in rat neutrophils.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed2005

Anaesthetic sensitivity of fMLP-induced cell signalling in Xenopus oocytes.

FMLP stimulation of Xenopus oocytes expressing fMLP receptors leads to a concentration-dependent biphasic inward current. To identify the evolution of these currents we have examined the effects of blocking various cell signalling pathways. In addition we have analysed the effects of three intravenous anaesthetics on these fMLP-induced currents. Xenopus oocytes were microinjected with cRNA encoding the fMLP receptor and fMLP-stimulated (100 nM) currents measured, using two-electrode voltage-clamp (-70 mV), before and after injection of heparin (120 ng ml-1), wortmannin (1 microM), U73122 (5 microM) or buffer. Concentration-response curves were established for the action on fMLP-stimulated currents of thiopentone (5-500 microM), methohexitone (0.2-200 microM) and propofol (0.5-500 microM). Heparin significantly enhanced the fast current (p<0.05). Wortmannin had no effect on either current. U73122 inhibited only the slow current (p<0.05). All anaesthetics inhibited both currents, with the maximum inhibition for the fast/slow currents 70%/100%, 60%/60% and 100%/100% for thiopentone (IC50 147/120 microM), methohexitone (IC50 4.7/2.2 microM) and propofol (IC50 33/8 microM), respectively. We suggest (a) the slow current arises via the PLC/PKC pathway because it is reduced by the PLC inhibitor U73122, (b) the PI3K- and PLD-mediated pathways are not involved because wortmannin had no effect and (c) activation of the two conductance channels must be different because U73122 reduced the slow but not the fast current. Since both currents are decreased by all three anaesthetics, their inhibition might be mediated through an action at the agonist/receptor, although, since the slow current is consistently more sensitive than the fast, there may be additionally an action on cell signalling.

Anesthetics, Intravenous↗
PubMedDate not supplied

Role of X-linked inhibitor of apoptosis protein in chemoresistance in ovarian cancer: possible involvement of the phosphoinositide-3 kinase/Akt pathway.

Although cisplatin derivatives are first-line chemotherapeutic agents for the treatment of epithelial ovarian cancer, chemoresistance remains a major hurdle to successful therapy and the molecular mechanisms involved are poorly understood. Apoptosis is the cellular underpinning of cisplatin-induced cell death, which is associated with expression of specific "death" genes and down-regulation of "survival" counterparts. The X-linked inhibitor of apoptosis proteins (Xiap), an intracellular anti-apoptotic protein, plays a key role in cell survival by modulating death signaling pathways and is a determinant of cisplatin resistance in ovarian cancer cells in vitro. This review focuses on the role of Xiap and its interactions with the phosphoinositide-3 kinase (PI3K)/Akt cell survival pathway in conferring resistance of ovarian cancer cells to chemotherapeutic agents and discusses potential therapeutic strategies in overcoming chemoresistant ovarian cancer.

Apoptosis↗