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PubMed2004

Targeted expression of the class II phosphoinositide 3-kinase in Drosophila melanogaster reveals lipid kinase-dependent effects on patterning and interactions with receptor signaling pathways.

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.

Animals↗
PubMed2003

Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells.

BACKGROUND AND AIMS: Determination of intracellular signalling pathways that mediate intestinal epithelial proliferation is fundamental to the understanding of the integrity and function of the intestinal tract under normal and diseased conditions. The phosphoinositide 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in cell proliferation and differentiation. In this study, we assessed the role of PI3K/Akt in transduction of proliferative signals in intestinal epithelial cells. METHODS: A rat intestinal epithelial (RIE) cell line and human colorectal cancer HCA-7 and LS-174 cell lines served as in vitro models. The Balb/cJ mouse was the in vivo model. RESULTS: PI3K activation was critical for G1 cell cycle progression of intestinal epithelial cells. Ectopic expression of either active p110alpha or Akt-1 increased RIE cell proliferation. In vivo experiments demonstrated that PI3K activation was closely associated with the proliferative activity of intestinal mucosa. Treatment of mice with PI3K inhibitors blocked induction of PI3K activity and attenuated intestinal mucosal proliferation associated with oral intake. Epidermal growth factor and transforming growth factor alpha stimulated PI3K activation which was required for growth factor induced expression of cyclin D1. CONCLUSIONS: The PI3K/Akt pathway transduces mitogenic signals from growth factor receptors to the cell cycle machinery and plays a critical role in regulation of intestinal epithelial proliferation.

Animals↗
PubMed2006

Neuregulin-dependent protein synthesis in C2C12 myotubes and rat diaphragm muscle.

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [(3)H]phenylalanine into the protein pool) were investigated in differentiated C(2)C(12) myotubes and rat diaphragm muscle (DIAm). In C(2)C(12) myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased approximately 30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [(3)H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

Androstadienes↗
PubMed1996

Mitogenic inhibition by phorbol esters is associated with decreased phosphatidylinositol-3 kinase activation.

In contrast to their role as potent tumor promoters, phorbol esters can cause inhibition of cell growth. Because the effect of phorbol esters occurs through activation of protein kinase C (PKC) and because activated PKC is translocated to the membrane placing it in a position to act on the intracellular portion of the growth factor receptor, we asked whether this inhibitory effect is mediated through the action of phorbol 12-myristate 13-acetate (PMA) on receptor association with the signal transfer proteins. When added to rat vascular smooth muscle (VSM) cells concurrently with basic fibroblast growth factor (bFGF), PMA at 100 ng/ml completely inhibits bFGF-stimulated DNA synthesis. Under the same growth-inhibitory conditions of PMA addition, aggregation of phosphatidylinositol 3-kinase (PI3K) to the fibroblast growth factor receptor and tyrosine phosphorylation of the 85-kDa regulatory component of the signal transfer protein PI3K are reduced by 94 and 79%, respectively. PI3K catalytic activity, as measured by conversion of phosphatidylinositol to phosphatidylinositol 3-phosphate, is decreased 88% by PMA addition. This effect is not specific to PI3K, since aggregation of phospholipase C-gamma 1 to the activated bFGF receptor is also decreased by PMA treatment. In addition, the PI3K inhibitor wortmannin markedly attenuates bFGF-stimulated VSM cell growth in a dose-dependent manner. These data suggest that the site of growth inhibition by PMA in VSM cells lies upstream of signal transfer particle aggregation and that such growth arrest may be mediated through inhibition of activation of PI3K.

Animals↗
PubMed2005

Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute beta-adrenergic stimulation.

Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development.

Adrenergic beta-Agonists↗
PubMed2006

Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells.

Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with TNF-alpha, ANG II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with wortmannin or LY-294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly, overexpression of a dominant-negative mutant of PI3K or Akt increased TNF-alpha- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and posttranscriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF-alpha and insulin is mediated, in part, by ERK and p38 MAPK. PI3K inhibitors augmented TNF-alpha- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which is known to activate PI3K/Akt, blocks TNF-alpha- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF-alpha- and insulin-induced PAI-1 expression. These findings indicate that the PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors that activate the PI3K/Akt pathway in ECs may have therapeutic benefits for atherothrombotic vascular disease.

Androstadienes↗
PubMed1999

Wortmannin inhibits insulin-stimulated activation of protein phosphatase 1 in rat cardiomyocytes.

A major function of insulin in target tissues is the activation of glycogen synthase. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the insulin-induced activation of glycogen synthase, although the true function of this enzyme remains unclear. Data presented here demonstrate that the PI3K inhibitors wortmannin and LY-294002 block the insulin-stimulated activation of protein phosphatase 1 (PP1) in rat ventricular cardiomyocytes. This loss of phosphatase activation mimics that seen in diabetic cardiomyocytes, in which insulin stimulation fails to activate both PP1 and glycogen synthase. Interestingly, in diabetic cells, insulin stimulated PI3K activity to 300% of that in untreated controls, whereas this activity was increased by only 77% in normal cells. PI3K protein levels, however, were similar in normal and diabetic cells. Our results indicate that PI3K is involved in the stimulation of glycogen synthase activity by insulin through the regulation of PP1. The inability of insulin to stimulate phosphatase activity in diabetic cells, despite a significant increase in PI3K activity, suggests a defect in the insulin signaling pathway that contributes to the pathology of insulin-dependent diabetes.

Androstadienes↗
PubMed1999

Vasopressin stimulates sodium transport in A6 cells via a phosphatidylinositide 3-kinase-dependent pathway.

The enzyme phosphatidylinositide 3-kinase (PI3K) phosphorylates the D-3 position of the inositol ring of inositol phospholipids and produces 3-phosphorylated inositides. These novel second messengers are thought to mediate diverse cellular signaling functions. The fungal metabolite wortmannin covalently binds to PI3K and selectively inhibits its activity. The role of PI3K in basal and hormone-stimulated transepithelial sodium transport was examined using this specific inhibitor. Wortmannin, 50 nM, did not affect basal, aldosterone-stimulated, or insulin-stimulated transport in A6 cells. Wortmannin completely inhibits vasopressin stimulation of transport in these cells. Vasopressin stimulates PI3K activity in A6 cells. Vasopressin stimulation of transport is also blocked by 5 microM LY-294002, a second inhibitor of PI3K. One-hour preincubation with wortmannin blocked vasopressin stimulation of protein kinase A activity in the cells. Sodium transport responses to exogenous cAMP and forskolin, which directly activates adenylate cyclase, were not affected by wortmannin. These results indicate that wortmannin inhibits vasopressin stimulation of Na(+) transport at a site proximal to activation of adenylate cyclase. The results suggest that PI3K may be involved in receptor activation by vasopressin.

Androstadienes↗
PubMed2004

Roles of phosphatidylinositol 3'-kinase and mammalian target of rapamycin/p70 ribosomal protein S6 kinase in K-Ras-mediated transformation of intestinal epithelial cells.

Phosphatidylinositol 3'-kinase (PI3K) activity is required for Ras- mediated transformation of intestinal epithelial cells (IECs). The mammalian target of rapamycin (mTOR) and its downstream pathways control the translation of specific mRNAs that are required for cell proliferation and transformation. Here, we elucidated the roles of PI3K and mTOR in K-Ras-mediated transformation of IECs (IEC-6). Induction of K-Ras activated PI3K and mTOR in IECs. p70 ribosomal protein S6 kinase activity was induced by K-Ras in a PI3K- and mTOR-dependent manner. K-Ras did not significantly alter the phosphorylation of eukaryotic initiation factor 4E-binding protein 1. Treatment with either LY-294002 or rapamycin inhibited IEC proliferation and resulted in G(1) growth arrest. However, it was noted that inhibition of mTOR enhanced K-Ras-mediated morphological transformation and increased invasiveness of IECs in a mitogen-activated protein/extracellular signal-regulated kinase-dependent manner. Furthermore, inhibition of PI3K or mTOR impaired the growth of an array of colon cancer cells. Spindle transformation, reduced E-cadherin, and increased invasiveness were observed in LY-294002-treated Moser cells. Thus, our results suggest that K-Ras-mediated transformation of IECs involves activation of the PI3K/mTOR pathway. Inhibition of PI3K/mTOR activity leads to G(1) growth arrest of transformed IECs. On the other hand, inhibition of PI3K or mTOR may induce the epithelial to mesenchymal transdifferentiation of IECs under certain circumstances.

Cell Differentiation↗
PubMed2004

Phosphatidylinositol 3'-kinase activation leads to multidrug resistance protein-1 expression and subsequent chemoresistance in advanced prostate cancer cells.

The development of androgen-independent growth in advanced carcinoma of the prostate (CaP) is associated with poor prognosis and few therapeutic options. Chemotherapeutic drugs offer the afflicted patient palliative benefits, but these are short-lived because of the chemoresistant nature of hormone-refractory prostate cancer. Given the high percentage of CaP patients with mutations in the PTEN tumor suppressor gene, we sought to determine the involvement of the phosphatidylinositol 3'-kinase (PI3K) cascade in the development of CaP drug resistance. PTEN-negative PC3 cells were observed to have increased resistance to both doxorubicin and paclitaxel when compared with PTEN-positive DU145 cells. Furthermore, modulation of PI3K activity with the use of constitutively active and dominant-negative inhibitors was found to affect the ability to CaP cells to respond to chemotoxic treatments. Additionally, inhibition of PI3K with a small molecular weight inhibitor (LY294002) was able to potentiate the antineoplastic activity of both doxorubicin and paclitaxel in CaP cells. Interestingly, multidrug resistance protein-1 (MRP-1) expression, but not MDR-1 (p-glycoprotein), was observed to be induced as a consequence of PI3K activation in these cell types. Inhibition of MRP-1 expression via siRNA was observed to synergistically sensitize CaP cells to chemotoxic drugs while having no appreciable effect on cell growth in the absence of these compounds. Taken together, these data suggest that PI3K activation can lead to the development of chemoresistant cells in prostatic carcinomas through the up-regulation of MRP-1. Thus, inhibition of PI3K activity with concomitant administration of chemotoxic compounds may prove beneficial in preventing the development of drug resistance in patients with hormone-refractory prostate cancer.

Base Sequence↗
PubMed2004

Adhesion-independent alpha6beta4 integrin clustering is mediated by phosphatidylinositol 3-kinase.

Clustering of cell-surface integrins is known to augment integrin-mediated signal transduction, but mechanisms of integrin clustering are poorly understood. Here we report that adhesion-independent clustering of alpha6beta4 integrin, known to be important in mediating tumor cell motility, is driven by phosphatidylinositol 3-kinase (PI3K) but does not require activation of the PI3K-Akt pathway. We observed clustering of alpha6beta4 in breast carcinoma cells after adhesion-independent cross-linking of the beta4 integrin subunit. Clustering was significantly blocked when cross-linking was performed in the presence of PI3K inhibitors LY294002 and wortmannin. In contrast, no significant inhibition of clustering was observed with protein kinase C inhibitor GF109203X, rapamycin, or heparin. Although alpha6beta4 clustering was blocked by PI3K inhibitors, clustering was not associated with increased PI3K lipid kinase activity or increased phosphorylation of Akt. A novel role for PI3K in alpha6beta4 integrin clustering is proposed.

Breast Neoplasms↗
PubMed2006

Activating transcription factor 3 and early growth response 1 are the novel targets of LY294002 in a phosphatidylinositol 3-kinase-independent pathway.

LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, has been widely used to study the function of PI3K in cellular responses. Based on its inhibitory effect on PI3K, LY294002 has been shown to exert antitumorigenic effect in vivo and in vitro. Here, we report that LY294002 alters early growth response 1 (EGR-1) phosphorylation and subsequently enhances activating transcription factor 3 (ATF3) expression independently of PI3K inhibition. This pathway may be, in part, responsible for the antitumorigenic effect of LY294002 in human colorectal cancer cells. ATF3 expression was increased by LY294002, followed by the induction of apoptosis in several colorectal cancer cell lines. This is consistent with results showing that the down-regulation of the ATF3 gene by small interfering RNA suppressed LY294002-induced apoptosis in HCT-116 cells. On the other hand, ATF3 expression was not affected by another PI3K inhibitor, wortmannin, as well as phosphatase and tensin homologue or dominant-negative Akt overexpression. We also found that LY294002 increases ATF3 promoter activity and the transactivation is partly mediated by a GC-rich sequence located in the promoter. EGR-1 binds to the ATF3 promoter as assessed by gel shift assay. Furthermore, phosphorylated EGR-1 was highly increased in LY294002-treated cells, indicating that EGR-1 phosphorylation induced by LY294002 may facilitate ATF3 transactivation. Our data suggest that EGR-1 acts as a mediator in LY294002-induced ATF3 expression via a PI3K-independent pathway. ATF3 and EGR-1 may provide a novel explanation for the antitumorigenic properties of LY294002 in human colorectal cancer cells.

Activating Transcription Factor 3↗
PubMed2006

Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma.

Amplification of MYCN occurs commonly in neuroblastoma. We report that phosphatidylinositol 3-kinase (PI3K) inhibition in murine neuroblastoma (driven by a tyrosine hydroxylase-MYCN transgene) led to decreased tumor mass and decreased levels of Mycn protein without affecting levels of MYCN mRNA. Consistent with these observations, PI3K inhibition in MYCN-amplified human neuroblastoma cell lines resulted in decreased levels of Mycn protein without affecting levels of MYCN mRNA and caused decreased proliferation and increased apoptosis. To clarify the importance of Mycn as a target of broad-spectrum PI3K inhibitors, we transduced wild-type N-myc and N-myc mutants lacking glycogen synthase kinase 3beta phosphorylation sites into human neuroblastoma cells with no endogenous expression of myc. In contrast to wild-type N-myc, the phosphorylation-defective mutant proteins were stabilized and were resistant to the antiproliferative effects of PI3K inhibition. Our results show the importance of Mycn as a therapeutic target in established tumors in vivo, offer a mechanistic rationale to test PI3K inhibitors in MYCN-amplified neuroblastoma, and represent a therapeutic approach applicable to a broad range of cancers in which transcription factors are stabilized through a PI3K-dependent mechanism.

Animals↗
PubMed2006

Role of focal adhesion kinase and phosphatidylinositol 3'-kinase in integrin fibronectin receptor-mediated, matrix metalloproteinase-1-dependent invasion by metastatic prostate cancer cells.

alpha(5)beta(1) Integrin interacts with the PHSRN sequence of plasma fibronectin, causing constitutive invasion by human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as in vitro invasion substrates. Immunoassays were employed to dissect the roles of focal adhesion kinase (FAK), phosphatidylinositol 3'-kinase (PI3K), and protein kinase Cdelta (PKC delta) in alpha(5)beta(1)-mediated, matrix metalloproteinase-1 (MMP-1)-dependent invasion by metastatic human DU 145 prostate cancer cells. We found that a peptide composed of the PHSRN sequence induced rapid FAK phosphorylation at Tyr(397) (Y397), a site whose phosphorylation is associated with kinase activation. The technique of RNA silencing [small interfering RNA (siRNA)] confirmed the role of FAK in PHSRN-induced invasion. PHSRN also induced the association of the p85-regulatory subunit of PI3K with FAK at a time corresponding to FAK phosphorylation and activation, and maximal PI3K activity occurred at this same time. The necessity of PI3K activity in both PHSRN-induced invasion and MMP-1 expression was confirmed by using specific PI3K inhibitors. By employing a specific inhibitor, Rottlerin, and by using siRNA, we also found that PKC delta, a PI3K substrate found in focal adhesions, functions in PHSRN-induced invasion. In addition, the induction of MMP-1 in PHSRN-treated DU 145 cells was shown by immunoblotting, and the role of MMP-1 in PHSRN-induced invasion was confirmed by the use of blocking anti-MMP-1 monoclonal antibody. Finally, a close temporal correspondence was observed between PHSRN-induced invasion and PHSRN-induced MMP-1 activity in DU 145 cells.

Adenocarcinoma↗
PubMed2005

Different activation of mitogen-activated protein kinase and Akt signaling is associated with aggressive phenotype of human meningiomas.

PURPOSE: Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. EXPERIMENTAL DESIGN: Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. RESULTS: Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. CONCLUSIONS: Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

Aged↗
PubMed2006

Delineating the mechanism by which selenium deactivates Akt in prostate cancer cells.

The up-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is prevalent in many cancers. This phenomenon makes PI3K and Akt fruitful targets for cancer therapy and/or prevention because they are mediators of cell survival signaling. Although the suppression of phospho-Akt by selenium has been reported previously, little information is available on whether selenium modulates primarily the PI3K-phosphoinositide-dependent kinase 1 (PDK1) side of Akt phosphorylation or the phosphatase side of Akt dephosphorylation. The present study was aimed at addressing these questions in PC-3 prostate cancer cells which are phosphatase and tensin homologue-null. Our results showed that selenium decreased Akt phosphorylation at Thr308 (by PDK1) and Ser473 (by an unidentified kinase); the Thr308 site was more sensitive to selenium inhibition than the Ser473 site. The protein levels of PI3K and phospho-PDK1 were not affected by selenium. However, the activity of PI3K was reduced by 30% in selenium-treated cells, thus discouraging the recruitment of PDK1 and Akt to the membrane due to low phosphatidylinositol-3,4,5-trisphosphate formation by PI3K. Consistent with the above interpretation, the membrane localization of PDK1 and Akt was significantly diminished as shown by Western blotting. In the presence of a calcium chelator or a specific inhibitor of calcineurin (a calcium-dependent phosphatase), the suppressive effect of selenium on phospho-Akt(Ser473) was greatly reduced. The finding suggests that selenium-mediated dephosphorylation of Akt via calcineurin is likely to be an additional mechanism in regulating the status of phospho-Akt.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Phosphatidylinositol 3-kinase in the G protein-coupled receptor-induced chemokinesis and chemotaxis of MDA-MB-468 breast carcinoma cells: a comparison with leukocytes.

The polarization of tumor cells and leukocytes into a front end and a rear end is a crucial prerequisite for their autonomous, directed movement. Phosphatidylinositol 3-kinase (PI3K) is assumed to play an important role in this polarization process, whereas the results obtained with different cell types and different migration assays widely vary. Thus, we conducted a comparative study on the role of the PI3K in the locomotor activity and directionality of the migration of tumor cells on the example of MDA-MB-468 breast carcinoma cells in comparison with CTLs and neutrophil granulocytes. We used our well-established, collagen-based, three-dimensional migration assay for the investigation of the chemokinesis and chemotaxis of these cells. Our results show that the role of the PI3K in the regulation of migratory activity is distinct between the investigated cell types: the migration of CTLs and MDA-MB-468 cells was impaired by the inhibition of the PI3K with wortmannin, whereas neutrophil granulocytes were only slightly affected. However, neither cell type was impaired in the ability to respond chemotactically to gradients of ligands to G protein-coupled receptors. Thus, the PI3K contributes to the regulation of migratory activity but not to the directionality of migration of MDA-MB-468 breast carcinoma cells. As a further conclusion with regard to cancer treatment, the PI3K is not a suitable target for the inhibition of metastasis formation, because the migration of leukocytes is also affected, which leads to a dysfunction of the immune defense.

Breast Neoplasms↗
PubMed2005

Phosphatidylinositide 3-kinase is important in late-stage fibroblast growth factor-1-mediated angiogenesis in vivo.

We previously reported that overexpression of a secreted version of fibroblast growth factor-1 (sp-FGF-1) has the ability to induce angiogenesis in the chicken chorioallantoic membrane (CAM). In our current study, we examine the effects of sp-FGF-1 through a time course analysis of angiogenesis in the chicken CAM on days 3, 4, and 5 after gene transfection. Significant angiogenesis was observed on days 4 and 5 after gene transfection in the CAM assay. To evaluate the role of phosphatidylinositide 3-kinase (PI3K) signaling in sp-FGF-1-induced angiogenesis, we analyzed mRNA expression levels of PI3K and protein activity through its immediate downstream target, AKT-1. We found upregulation of both PI3K and AKT mRNA expression levels in day 5 sp-FGF-1 versus day 5 vector control-transfected CAMs. Furthermore, by blocking PI3K phosphorylation using the specific inhibitor, LY294002, we found that downstream phosphorylation of AKT-1 was inhibited. More importantly, the blockade of the PI3K pathway via LY294002 in sp-FGF-1-transfected CAMs significantly inhibited angiogenesis. These results further elucidate the molecular mechanisms of the sp-FGF-1 signaling pathway and it underscores the importance of PI3K signaling in FGF-1-stimulated angiogenesis in vivo. It also provides a basis for the role of sp-FGF-1 in the development of therapeutic treatments to combat vascular insufficiencies and angiogenesis-dependent cancers.

Animals↗