DNA-PK phosphorylation of IGFBP-3 is required to Prevent Apoptosis in Retinal Endothelial cells cultured in High Glucose.

DNA-PK phosphorylation of IGFBP-3 is required to Prevent Apoptosis in Retinal Endothelial cells cultured in High Glucose.

Invest Ophthalmol Vis Sci. 2013 Apr 4;

Authors: Zhang Q, Steinle JJ

Abstract
PURPOSE: The goal of this study was to determine whether Compound 49b stimulates insulin-like growth factor binding protein-3 (IGFBP-3) activation in retinal endothelial cells (REC) through DNA-dependent protein kinase (DNA-PK). METHODS: REC were grown in normal glucose (5 mM) or high glucose medium (25 mM). Some cells were transfected with protein kinase A (PKA) siRNA, following treatment with 50 nM Compound 49b, a novel ?-adrenergic receptor agonist. Cell proteins were extracted and analyzed for DNA-PK expression by Western blotting. Additional cells were treated with or without NU7441 (a specific DNA-PK inhibitor) prior to Compound 49b treatment. Cell lysates were processed for IGFBP-3 ELISA analyses and Western blotting to measure casein kinase 2 (CK2). Immunoprecipitation for total and phospho-IGFBP-3 and cell death measurements were done after transfection with the S156A IGFBP-3 mutation (key phosphorylation site involved in DNA-PK) plasmid DNA. RESULTS: Compound 49b required DNA-PK to activate IGFBP-3 in REC. IGFBP-3 activation was significantly reduced following treatment with either the DNA-PK inhibitor or following transfection with the IGFBP-3 S156A mutant plasmid (p < 0.05). Significant increases in cell death were also observed in cells transfected with the IGFBP-3 S156A mutant plasmid (p < 0.05). Casein kinase levels were not altered after treatment with NU7741 or Compound 49b. CONCLUSIONS: Our finding suggested Compound 49b induces DNA-PK levels through PKA activity. DNA-PK is required for Compound 49b-induced activation of IGFBP-3, leading to inhibition of REC cell death.

PMID: 23557743 [PubMed - as supplied by publisher]

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EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors.

EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors.

Clin Cancer Res. 2013 Apr 3;

Authors: Bladt F, Faden B, Friese-Hamim M, Knuehl C, Wilm C, Frittschen C, Graedler U, Meyring M, Dorsch D, Jaehrling F, Pehl U, Stieber F, Schadt O, Blaukat A

Abstract
PURPOSE: The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds, developed to selectively inhibit the c-Met receptor in anti-tumor therapeutic interventions. EXPERIMENTAL DESIGN: The pharmacologic properties, c-Met inhibitory activity, and anti-tumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models. RESULTS: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent (inhibitory 50% concentration [IC50], 3 nM and 9 nM, respectively) and highly selective, when compared to their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after >3 weeks of treatment. CONCLUSIONS: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anti-cancer strategies.

PMID: 23553846 [PubMed - as supplied by publisher]

c-met inhibitors zm-447439 rad001

DNA-PK phosphorylation of IGFBP-3 is required to Prevent Apoptosis in Retinal Endothelial cells cultured in High Glucose.

DNA-PK phosphorylation of IGFBP-3 is required to Prevent Apoptosis in Retinal Endothelial cells cultured in High Glucose.

Invest Ophthalmol Vis Sci. 2013 Apr 4;

Authors: Zhang Q, Steinle JJ

Abstract
PURPOSE: The goal of this study was to determine whether Compound 49b stimulates insulin-like growth factor binding protein-3 (IGFBP-3) activation in retinal endothelial cells (REC) through DNA-dependent protein kinase (DNA-PK). METHODS: REC were grown in normal glucose (5 mM) or high glucose medium (25 mM). Some cells were transfected with protein kinase A (PKA) siRNA, following treatment with 50 nM Compound 49b, a novel ?-adrenergic receptor agonist. Cell proteins were extracted and analyzed for DNA-PK expression by Western blotting. Additional cells were treated with or without NU7441 (a specific DNA-PK inhibitor) prior to Compound 49b treatment. Cell lysates were processed for IGFBP-3 ELISA analyses and Western blotting to measure casein kinase 2 (CK2). Immunoprecipitation for total and phospho-IGFBP-3 and cell death measurements were done after transfection with the S156A IGFBP-3 mutation (key phosphorylation site involved in DNA-PK) plasmid DNA. RESULTS: Compound 49b required DNA-PK to activate IGFBP-3 in REC. IGFBP-3 activation was significantly reduced following treatment with either the DNA-PK inhibitor or following transfection with the IGFBP-3 S156A mutant plasmid (p < 0.05). Significant increases in cell death were also observed in cells transfected with the IGFBP-3 S156A mutant plasmid (p < 0.05). Casein kinase levels were not altered after treatment with NU7741 or Compound 49b. CONCLUSIONS: Our finding suggested Compound 49b induces DNA-PK levels through PKA activity. DNA-PK is required for Compound 49b-induced activation of IGFBP-3, leading to inhibition of REC cell death.

PMID: 23557743 [PubMed - as supplied by publisher]

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Association of a MET genetic variant with autism-associated maternal autoantibodies to fetal brain proteins and cytokine expression.

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Association of a MET genetic variant with autism-associated maternal autoantibodies to fetal brain proteins and cytokine expression.

Transl Psychiatry. 2011;1:e48

Authors: Heuer L, Braunschweig D, Ashwood P, Van de Water J, Campbell DB

Abstract
The contribution of peripheral immunity to autism spectrum disorders (ASDs) risk is debated and poorly understood. Some mothers of children with ASD have autoantibodies that react to fetal brain proteins, raising the possibility that a subset of ASD cases may be associated with a maternal antibody response during gestation. The mechanism by which the maternal immune system breaks tolerance has not been addressed. We hypothesized that the mechanism may involve decreased expression of the MET receptor tyrosine kinase, an ASD risk gene that also serves as a key negative regulator of immune responsiveness. In a sample of 365 mothers, including 202 mothers of children with ASD, the functional MET promoter variant rs1858830 C allele was strongly associated with the presence of an ASD-specific 37+73-kDa band pattern of maternal autoantibodies to fetal brain proteins (P=0.003). To determine the mechanism of this genetic association, we measured MET protein and cytokine production in freshly prepared peripheral blood mononuclear cells from 76 mothers of ASD and typically developing children. The MET rs1858830 C allele was significantly associated with MET protein expression (P=0.025). Moreover, decreased expression of the regulatory cytokine IL-10 was associated with both the MET gene C allele (P=0.001) and reduced MET protein levels (P=0.002). These results indicate genetic distinction among mothers who produce ASD-associated antibodies to fetal brain proteins, and suggest a potential mechanism for how a genetically determined decrease in MET protein production may lead to a reduction in immune regulation.

PMID: 22833194 [PubMed - indexed for MEDLINE]

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In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells.

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells.

Transl Oncol. 2013 Apr;6(2):104-11

Authors: Rath P, Lal B, Ajala O, Li Y, Xia S, Kim J, Laterra J

Abstract
Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.

PMID: 23556031 [PubMed - in process]

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Monoterpenoid indole alkaloids from Alstonia yunnanensis and their cytotoxic and anti-inflammatory activities.

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Monoterpenoid indole alkaloids from Alstonia yunnanensis and their cytotoxic and anti-inflammatory activities.

Molecules. 2012;17(11):13631-41

Authors: Cao P, Liang Y, Gao X, Li XM, Song ZQ, Liang G

Abstract
The 80% ethanol extract of Alstonia yunnanensis afforded five new monoterpenoid indole alkaloids: 11-hydroxy-6,7-epoxy-8-oxo-vincadifformine (1), 14-chloro-15-hydroxy- vincadifformine (2), perakine N(4)-oxide (3), raucaffrinoline N(4)-oxide (4), and vinorine N(1),N(4)-dioxide (5), together with three known compounds: 11-methoxy-6,7-epoxy-8-oxo- vincadifformine (6), vinorine N(4)-oxide (7) and vinorine (8). The structures of the isolated compounds were established based on 1D and 2D (1H-1H-COSY, HMQC, HMBC, and ROESY) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic potential against seven tumor cell lines and anti-inflammatory activities. Compounds 3, 4 and 7 exhibited weak cytotoxicity against the tested cell lines and selective inhibition of Cox-2 (> 85%).

PMID: 23159924 [PubMed - indexed for MEDLINE]

chir-258 dovitinib dna-pk

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells.

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells.

Transl Oncol. 2013 Apr;6(2):104-11

Authors: Rath P, Lal B, Ajala O, Li Y, Xia S, Kim J, Laterra J

Abstract
Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.

PMID: 23556031 [PubMed - in process]

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