epidermal-growth-factor and pyrazolanthrone

Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration.. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting.. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720.. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1.

Topics: Animals; Anthracenes; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Flavonoids; Heterocyclic Compounds, 4 or More Rings; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Resveratrol; rho-Associated Kinases; Sirtuin 1; Stilbenes

The objective of this work was to study the effect of epidermal growth factor (EGF) induced secretions of angiogenesis factors in adipose-derived stem cells (ADSCs) and the involvement of mitogen-activated protein kinases (MAPK). ADSCs were cultured and ELISA assays were performed to quantify the vascular endothelial growth factor, the hepatocyte growth factor, and the stromal derived factor-1 in ADSC-conditioned medium before and after EGF treatments and after pharmacological inhibition of MAPKs with PD98059, SB203580, and SP600125. The tube formation assay was used to test the effects of EGF treated and inhibitor treated ADSCs on the human umbilical vein endothelial cells (HUVECs) tube formation. Liposuction was applied and ADSCs were cultured successfully. The ADSCs released a variety of angiogenic factors, with the EGF treatments enhancing secretions and promoting the HUVEC tube formation. The MAPK inhibitors PD98059 and SP600125 increased the paracrine to promote tubular formation, while the SB203580 played an opposite role. In conclusion, (1) the in vitro cultured ADSCs secrete various angiogenic factors and the EGF amplifies the secretion and can enhance the ADSCs on the HUVEC tube formation. (2) ERK1/2 and JNK pathway may be involved in the enhanced secretion capacity of ADSCs while the p38 pathway may exert an opposite effect.

Topics: Adipose Tissue; Adult; Anthracenes; Chemokine CXCL12; Culture Media, Conditioned; Epidermal Growth Factor; Female; Flavonoids; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Imidazoles; Lipectomy; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Primary Cell Culture; Protein Kinase Inhibitors; Pyridines; Signal Transduction; Stem Cells; Vascular Endothelial Growth Factor A

Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma.. Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown.. EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α.. EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

Topics: Animals; Anthracenes; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Autocrine Communication; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cetuximab; Endoribonucleases; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Gene Expression; Glioma; Humans; JNK Mitogen-Activated Protein Kinases; Mice; Protein Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The purpose of this research is to study the effect of genistein on cytokines or growth factor-induced proliferation and transformation phenotype of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were primarily cultured. With respective stimulation of IL-1β, TNF-α, and EGF, genistein was applied to elucidate its effect on synoviocytes' growth number, cell proliferation assay, cell cycle using cell counts, (3)H-TdR incorporation and flow cytometry, the colony numbers under anchorage-independent condition, and the expression of MMP-2 and MMP-9 in synovial fibroblasts. EGF, IL-1β, and TNF-α increased (3)H incorporation in RA-FLS, respectively. EGF augmented clone numbers of RA-FLS under anchorage-independent condition and not IL-1β and TNF-α. Genistein had an inhibitory role on cell number and (3)H-TdR incorporation of RA-FLS stimulated with IL-1β, TNF-α and EGF; genistein arrested the cell cycle at G(1) restriction point; genistein decreased colony numbers under anchorage-independent condition stimulated by EGF in serum condition. IL-1β or TNF-α increased expression of MMP-9 and MMP-2 in rheumatoid synoviocytes; EGF stimulated expression of MMP-9 but not of MMP-2; genistein suppressed production of MMP-9 more than MMP-2 induced by IL-1β or TNF-α; rMMP-9, rMMP-2, or their inhibitors had no effect on the (3)H-TdR incorporation of synovial cells. Erk1/2 inhibitor (PD098 059) had obvious inhibitory effect on the (3)H incorporation induced by TNF-α or IL-1β; inhibitors of JNK (SP600 125) had no significant effect on the (3)H incorporation. While pretreatment with PD098059 had no marked inhibitory effect on MMP-9 expression induced by TNF-α or IL-1β, SP600125 decreased significantly the MMP-9 expression induced by TNF-α or IL-1β. Neither PD098059 nor SP600 125 could inhibit the MMP-2 expression induced by TNF-α or IL-1β. Genistein inhibited IL-1β, TNF-α or EGF-induced proliferation and MMP-9 expression in fibroblast-like synoviocytes of rheumatoid arthritis; the proliferation of RA-FLS was mediated by Erk1/2 but not JNK activation, while JNK activation was involved in the signal transduction pathway leading to MMP-9 expression in rheumatoid synoviocytes.

Topics: Anthracenes; Arthritis, Rheumatoid; Cell Cycle; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Flavonoids; G1 Phase Cell Cycle Checkpoints; Genistein; Humans; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Synovial Membrane; Tumor Necrosis Factor-alpha

Although it has been suggested that smad7 blocks downstream signaling of TGF-β, the role of smad7 in the EGF signaling pathway has not been fully elucidated. We determined the effect of smad7 on EGF-induced MMP-9 expression in SKBR3 breast cancer cells. The expression of smad7 and MMP-9 was increased by EGF or TGF-β1, respectively, and further increased by EGF and TGF-β1 co-treatment. EGF induced the phosphorylation of EGFR, smad3, ERK, and JNK, and MMP-9 expression was decreased by the EGFR inhibitor, AG1478. In addition, EGF-induced MMP-9 expression was inhibited by UO126 (a MEK1/2 inhibitor) or SIS3 (a smad3 inhibitor), but not by SP600125 (a JNK inhibitor). Interestingly, EGF-induced smad3 phosphorylation was completely blocked by smad7 over-expression, but not the phosphorylation of ERK and JNK. EGF- or TGF-β1-induced MMP-9 expression was completely decreased by adenoviral-smad7 (Ad-smad7) over-expression. We also investigated the role of smad3 on EGF-induced MMP-9 expression and showed that EGF-induced MMP-9 expression was decreased by smad3 siRNA transfection, whereas EGF-induced MMP-9 expression was further increased by smad3 over-expression, as expected. This study showed that EGF-induced smad3 phosphorylation mediates the induction of MMP-9, whereas smad7 inhibits TGF-β1 as well as the EGF signaling pathway in SKBR3 cells.

Topics: Anthracenes; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 9; Nitriles; Phosphorylation; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta1

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.

Topics: Acetaldehyde; Actins; Adherens Junctions; Anthracenes; beta Catenin; Butadienes; Caco-2 Cells; Cadherins; Claudin-3; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Inulin; Membrane Proteins; Nitriles; Occludin; Permeability; Phosphoproteins; Pyridines; Tight Junctions; Zonula Occludens-1 Protein

We have recently demonstrated that tissue inhibitor of metalloproteinase-3 (TIMP-3) decreases neonatal cardiomyocyte proliferation (Hammoud L, Xiang F, Lu X, Brunner F, Leco K, Feng Q. Cardiovasc Res 75: 359-368, 2007). The aim of the present study was to delineate a pathway through which TIMP-3 exerts its antiproliferative effect. Experiments were conducted on neonatal cardiomyocyte cultures and heart tissues isolated from wild-type (WT) and TIMP-3(-/-) mice. Deficiency in TIMP-3 decreased p27 expression and increased cardiomyocyte proliferation in cardiomyocytes and neonatal hearts. A TIMP-3/epidermal growth factor (EGF) receptor (EGFR)/c-Jun NH(2)-terminal kinase (JNK)/SP-1/p27 pathway was investigated. JNK phosphorylation and EGFR protein levels were increased in TIMP-3(-/-) cardiomyocytes and heart tissues. Treatment with recombinant TIMP-3 decreased JNK phosphorylation and EGFR expression/phosphorylation. Inhibition of JNK activity using SP-600125 decreased SP-1 phosphorylation, increased p27 expression, and decreased cardiomyocyte proliferation. Furthermore, treatment with the EGFR specific inhibitor PD-168393 or the EGF-neutralizing antibody decreased cardiomyocyte proliferation as well as phosphorylation of JNK and SP-1 in both WT and TIMP-3(-/-) cardiomyocytes. We conclude that TIMP-3 inhibits neonatal mouse cardiomyocyte proliferation by upregulating p27 expression. The effects of TIMP-3 are mediated via inhibition of EGFR expression/phosphorylation, and decreases in JNK and SP-1 signaling.

Topics: Animals; Animals, Newborn; Anthracenes; Cell Proliferation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Epidermal Growth Factor; ErbB Receptors; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Cardiac; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; Recombinant Proteins; Signal Transduction; Sp1 Transcription Factor; Tissue Inhibitor of Metalloproteinase-3

The kinase TAK1, a mitogen-activated protein kinase kinase kinase (MAP3K), has been widely accepted as a key kinase activating NF-kappaB and MAPKs in tumor necrosis factor alpha (TNF-alpha) signaling. We have recently reported that TAK1 regulates the transient phosphorylation and endocytosis of epidermal growth factor receptor (EGFR) in a tyrosine kinase activity-independent manner. In the present study, we found that Thr-669 in the juxtamembrane domain and Ser-1046/1047 in the carboxyl-terminal regulatory domain were transiently phosphorylated in response to TNF-alpha. Experiments using chemical inhibitors and small interfering RNA demonstrated that TNF-alpha-mediated phosphorylation of Thr-669 and Ser-1046/7 were differently regulated via TAK1-extracellular signal-regulated kinase (ERK) and TAK1-p38 pathways, respectively. In addition, p38, but not ERK, was involved in the endocytosis of EGFR. Surprisingly, modified EGFR was essential to prevent apoptotic cellular responses; however, the EGFR pathway was independent of the NF-kappaB antiapoptotic pathway. These results demonstrated that TAK1 controls two different signaling pathways, IkappaB kinase-NF-kappaB and MAPK-EGFR, leading to the survival of cells exposed to the death signal from the TNF-alpha receptor.

Topics: Anthracenes; Butadienes; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Imidazoles; Lactones; MAP Kinase Kinase Kinases; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Quinazolines; Resorcinols; RNA, Small Interfering; Serine; Signal Transduction; Threonine; Tumor Necrosis Factor-alpha

Epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression in squamous cell carcinomas is mediated through the extracellular signal-regulated kinase 1/2 and p38 pathways. Examination of a basaloid and a conventional oral squamous cell carcinoma cell line revealed that inhibition of c-Jun N-terminal kinase (JNK) with SP600125 increased EGF-induced (but not basal) COX-2 transcription 1.5-1.9-fold in extracellular signal-regulated kinase 1/2 and p38 pathway-dependent manners. Although JNK may phosphorylate the cyclosporine A-sensitive transcription factor, nuclear factor of activated T cells c3, it was seemingly not involved because cyclosporine A did not reduce EGF-induced COX-2 expression. Thus, JNK negatively regulated EGF-induced extracellular signal-regulated kinase 1/2 and/or p38-mediated COX-2 transcription, presumably through activating an unidentified phosphatase.

Topics: Anthracenes; Calcineurin Inhibitors; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclooxygenase 2; Cyclosporine; Dactinomycin; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; NFATC Transcription Factors; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Synthesis Inhibitors; Transcription, Genetic

In this study, we demonstrated that tyrosine phosphorylation of EGFR and the autocrine expression of uPA and HB-EGF depend on the activity of c-jun amino-terminal kinase (JNK) in human prostatic DU-145 cells. These cells overexpress EGFR and produce a high amount of uPA. Treatment with either SP600125, a specific chemical inhibitor of JNK, or the expression of a dominant-negative JNK form inhibited autocrine production of uPA and HB-EGF, which block EGFR phosphorylation and mitigates invasive capacity. Our data provided evidence that in DU-145 cells, the maintenance of the activation level of EGFR, which determines the cellular invasive potential, operates through an autocrine loop involving the JNK-dependent production of uPA and HB-EGF activity. Moreover, we found that exogenously added uPA stimulates autocrine production of HB-EGF, and that blocking HB-EGF activity curbed DU-145 cell invasive potential.

Topics: Anthracenes; Autocrine Communication; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Male; Neoplasm Invasiveness; Phosphorylation; Prostatic Neoplasms; Protein Kinase Inhibitors; Up-Regulation; Urokinase-Type Plasminogen Activator

The TGF-beta (transforming growth factor-beta) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor alpha-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-alpha and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-beta in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-beta, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TbetaR-I (TGF-beta receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-beta up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TbetaR-I-dependent manner. TGF-beta mediates activation of the nuclear factor-kappaB pathway, which is inhibited by DPI and is required for up-regulation of TGF-alpha and HB-EGF. In contrast, EGFR activation is not required for TGF-beta-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-beta in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-beta-treated cells.

Topics: Acetophenones; Animals; Anthracenes; Benzamides; Chromones; Dioxoles; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Heparin-binding EGF-like Growth Factor; Hepatocytes; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Morpholines; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 4; NADPH Oxidases; NF-kappa B; Onium Compounds; Peptides; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinazolines; rac1 GTP-Binding Protein; Rats; Reactive Oxygen Species; Transforming Growth Factor beta; Tyrphostins; Up-Regulation

Epidermal growth factor (EGF) reduces apoptosis in primary cytotrophoblast (CT) in culture through two separate pathways: the extracellular signal related kinase (ERK) 1/2 and phosphatidyl inositol 3-kinase (PI-3 kinase) paths. Whether other pathways are involved in survival signalling is unknown. We here show that the c-Jun NH2 terminal kinase (JNK) and the mitogen activated kinase (MAPK) p38 are also activated by EGF as seen by increases in JNK and p38 phosphorylation. However, inhibition of JNK phosphorylation with the specific inhibitor SP600125 increases apoptosis in a manner refractory to the addition of EGF but inhibition of p38 phosphorylation with its specific inhibitor SB 203580 does not increase apoptosis. EGF also activates sphingosine kinase-1 (SPHK-1), which converts sphingosine to sphingosine-1-phosphate, and its inhibition with dimethyl sphingosine (DMS) increased trophoblast death. Inhibition of SPHK-1 also did not affect EGF stimulated phosphorylation of PI-3 kinase, Akt, ERK1/2 or p38 but inhibition of PI-3 kinase with a specific inhibitor LY294002 partly (40%) inhibited the EGF-stimulated increase in SPHK-1 activity. We conclude that, in addition to the PI-3 kinase and ERK1/2 pathways, EGF acts through its receptor to stimulate JNK, p38 and SPHK-1 pathways, but that the JNK and SPHK-1, and not the p38, pathways are involved in suppressing apoptosis. This information provides evidence that EGF stimulates survival along multiple pathways that differ in trophoblast and other cell types.

Topics: Adaptor Proteins, Signal Transducing; Adult; Anthracenes; Apoptosis; Cell Culture Techniques; Chorionic Villi; Drug Combinations; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pregnancy; Pyridines; Signal Transduction; Trophoblasts

A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR-TGFalpha) results in a constitutive activation of MAPK-Elk1-AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR-Shc-MAPK-Elk1-AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involveme

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Anthracenes; Apoptosis; Cell Division; Cell-Free System; Culture Media, Serum-Free; DNA-Binding Proteins; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; ets-Domain Protein Elk-1; Flavonoids; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Phosphorylation; Plant Extracts; Prostatic Neoplasms; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Seeds; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured; Vitis

The biological function of full-length amyloid-beta protein precursor (AbetaPP), the precursor of Abeta, is not fully understood. Multiple laboratories have reported that antibody binding to cell surface AbetaPP causes neuronal cell death. Here we examined whether induced dimerization of the cytoplasmic domain of AbetaPP (AbetaPPCD) triggers neuronal cell death. In neurohybrid cells expressing fusion constructs of the epidermal growth factor (EGF) receptor with AbetaPPCD (EGFR/AbetaPP hybrids), EGF drastically enhanced neuronal cell death in a manner sensitive to acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-aldehyde (Ac-DEVD-CHO; DEVD), GSH-ethyl ester (GEE), and pertussis toxin (PTX). Dominant-negative apoptosis signal-regulating kinase 1 (ASK1) blocked this neuronal cell death, but not alpha-synuclein-induced cell death. Constitutively active ASK1 (caASK1) caused DEVD/GEE-sensitive cell death in a manner resistant to PTX and sensitive to Humanin, which also suppressed neuronal cell death by EGFR/AbetaPP hybrid. ASK1 formed a complex with AbetaPPCD via JIP-1b, the c-Jun N-terminal kinase (JNK)-interacting protein. EGFR/AbetaPP hybrid-induced and caASK1-induced neuronal cell deaths were specifically blocked by SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), a specific JNK inhibitor. Combined with our earlier study, these data indicate that dimerization of AbetaPPCD triggers ASK1/JNK-mediated neuronal cell death. We also noticed a potential role of ASK1/JNK in sustaining the activity of this mechanism after initial activation by AbetaPP, which allows for the achievement of cell death by short-term anti-AbetaPP antibody treatment. Understanding the function of AbetaPPCD and its downstream pathway should lead to effective anti-Alzheimer's disease therapeutics.

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Anthracenes; Carrier Proteins; Dimerization; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; Hybrid Cells; Imidazoles; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 5; MAP Kinase Kinase Kinases; Mice; Mitogen-Activated Protein Kinases; Neurons; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Structure, Tertiary; Proteins; Pyridines; Rats; Signal Transduction