17 research outputs found

    PKC COME NUOVO TARGET MOLECOLARE NEL CONTROLLO DELLA PROLIFERAZIONE CELLULARE DEI TUMORI NEUROENDOCRINI

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    Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures, in BON1 cell line, in human medullary thyroid carcinoma (MTC) primary culture and in TT cell line. We found that PKC inhibition reduces cell proliferation by inducing caspase mediated apoptosis and blocks the stimulatory effects of IGF-I on CT secretion in MTC, and CgA secretion in PNN. Enzastaurin reduces PKCβII(Thr500) phosphorylation, indicating a direct involvement of this isoform as well as phosphirylated levels of Akt(ser473) and GSK3β(Ser9), PKC pathway downstream targets and pharmacodynamic markers for PKC inhibition. PKCβII and PKCδ enzyme isoforms expression and localization were investigated. These data indicate that in vitro PKC is involved in the control of human PNN and human MTC proliferation and survival by modulating apoptosis, with a mechanism that implicates PKCβII inhibition and translocation in different subcellular compartments. In conclusion, targeting PKC may represent a useful therapeutic approach for controlling of neuroendocrine tumors proliferation

    Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors

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    Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures and in the human pancreatic endocrine cancer cell line, BON1. To this aim six human PNN dispersed in primary cultures and BON1 cells were treated without or with 1–10 μM Enzastaurin and/or 100 nM IGF1 in the presence or absence of serum. Cell viability and apoptosis were evaluated after 48–72 h; Chromogranin A (CgA) and/or insulin secretion was assessed after 6 h of incubation. PKC expression was investigated by immunofluorescence and western blot. We found that Enzastaurin significantly reduced human PNN primary culture cell viability, as well as CgA and insulin secretion. Moreover, in the BON1 cell line Enzastaurin inhibited cell proliferation at 5 and 10 μM by inducing caspase-mediated apoptosis, and reduced phosphorylation of glycogen synthetase kinase 3β (GSK3β) and of Akt, both downstream targets of PKC pathway and pharmacodynamic markers for Enzastaurin. In addition, Enzastaurin blocked the stimulatory effect of IGF1 on cell proliferation, and reduced CgA expression and secretion in BON1 cells. Two different PKC isoforms are expressed at different levels and have partially different subcellular localization in BON1 cells. In conclusion, Enzastaurin reduces cell proliferation by inducing apoptosis, with a mechanism likely involving GSK3β signaling, and inhibits secretory activity in PNNin vitromodels, suggesting that Enzastaurin might represent a possible medical treatment of human PNN

    Cyclo-Oxygenase 2 Modulates Chemoresistance in Breast Cancer Cells Involving NF-ÎşB

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    Background: Breast cancer cells can develop chemoresistance after prolonged exposure to cytotoxic drugs due to expression of the multi drug resistance (MDR) 1 gene. Type 2 cyclo-oxygenase (COX-2) inhibitors reverse the chemoresistance phenotype of a medullary thyroid carcinoma cell line, TT, and of a breast cancer cell line, MCF7, by inhibiting MDR1 expression and P-gp function

    Magmas expression in rat pituitary cell lines.

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    <p>(A) Expression of rat Magmas gene in GH1, GH3, GH4C1 and MMQ cell lines was determined by RT-qPCR and normalized to 18S rRNA as reference gene. Data are presented as fold change of mRNA levels of target gene (mean ± SE) in the cell lines vs. normal pituitary pool (NP), as detailed in Materials and Methods. *P < 0.05. (B) Expression of rat Magmas protein in GH3, GH4C1 and MMQ cell lines was determined by Western blot analysis and normalized to tubulin as housekeeping gene.</p

    Magmas-DD expression in GH4C1 cells.

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    <p>(A) GH4C1 cells were transfected with the pPTunerC Magmas-DD vector and treated without or with increasing concentrations of Shield1 (100-800 nM) for 12 h. Western blot analysis shows the transfected Magmas-DD protein (25 kDa), endogenous Magmas (13 kDa), as well as the internal control, actin (42 kDa). (B) GH4C1 cells were transfected with the pPTunerC Magmas-DD vector and treated without or with increasing concentrations of Shield1 (100-800 nM) for 12 h. Western blot analysis on mitochondria extracts shows the transfected Magmas-DD protein (25 kDa) as well as the internal control, Tomm (17 kDa).</p

    Magmas over-expression increases cell number and counteracts Staurosporine-induced apoptosis in GH4C1-M-DD cells.

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    <p>(A) GH4C1 were transfected or not with the pPTunerC Magmas- DD vector and then incubated in 96-well plates for 12, 24, 48 and 72 h in culture medium supplemented with or without 100 nM Staurosporine, in the absence or in the presence of 200 nM Shield1 concentrations. Cell count was then assessed as described in the Materials and Methods section. Data were evaluated in at least three independent experiments with four replicates each and are expressed as the mean value ± SE cell number/ml vs. control cells. *P < 0.05 vs. control cells. (B) GH4C1 were transfected or not with the pPTunerC Magmas-DD vector. Cell-cycle analysis was performed after treatment with or without 200 nM Shield 1. The graph shows representative data of three independent experiments, which were repeated three times. (C) GH4C1 were transfected or not with the pPTunerC Magmas- DD vector, incubated 24 h in culture medium supplemented with or without 100 nM Staurosporine, and analyzed for apoptosis after 24 hours. Cells were stained with FITC-conjugated annexin V and propidium iodide. Data were evaluated in at least three independent experiments with three replicates each and are expressed as % apoptosis. *P < 0.05 vs. control cells; #P<0.05 vs. GH4C1 cells treated with Staurosporine.</p

    Magmas over-expression increases cell viability and counteracts Staurosporine effects in GH4C1-M-DD cells.

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    <div><p>(A) GH4C1 were transfected or not with the pPTunerC Magmas- DD vector and then incubated in 96-well plates for 48 h in culture medium in the absence or in the presence of 200 nM Shield1 for 12 h before adding Staurosporine at increasing concentrations (from 20 to 300 nM). Data were evaluated in at least three independent experiments with four replicates each and are expressed as the mean value ± SE percent cell viability vs. control cells. *P < 0.05 vs. control cells. #P<0.05 vs. GH4C1 cells treated with Staurosporine.</p> <p>(B) GH4C1 were transfected or not with the pPTunerC Magmas- DD vector and then incubated in 96-well plates for 48 h in culture medium supplemented in the absence or in the presence of increasing Shield1 concentrations(from 100 nM to 400 nM) for 12 h before adding or not 100 nM Staurosporine. Cell viability were then assessed as described in the Materials and Methods section. Data were evaluated in at least three independent experiments with four replicates each and are expressed as the mean value ± SE percent cell viability vs. control cells. *P < 0.05 vs. control cells. #P<0.05 vs. GH4C1 cells treated with Staurosporine. Western blot analysis (middle panel) shows Magmas-DD protein expression levels, as well as the internal control, tubulin.</p></div

    Magmas over-expression inhibits caspase activation and cytochrome c release.

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    <div><p>A) GH4C1 were transfected or not with the pPTunerC Magmas-DD vector, incubated in 96-well plates overnight and then treated for 48 h with or without 100 nM Staurosporine in the presence or in the absence of 200 nM Shield1. Caspase 3/7 activation (upper panel) was assessed as described in the Materials and Methods section. Data were evaluated in at least six independent experiments with eight replicates each and are expressed as the mean value ± SE fold induction vs. control cells. *P< 0.05 vs. control; #P<0.05 vs. GH4C1 cells treated with Staurosporine. DNA fragmentation analysis (lower panel) was assessed as described in the Materials and Methods section. 1= GH4C1 control cells; 2= GH4C1-MDD cells with 200 nM Shield1 3 = GH4C1 cells treated with 100 nM Staurosporine; 4 = GH4C1 cells treated with 100 nM Staurosporine and with 200 nM Shield1; 5 = GH4C1-M-DD cells treated with 100 nM Staurosporine; 6 = GH4C1-MDD cells treated with 100 nM Staurosporine and with 200 nM Shield1.</p> <p>B) GH4C1-M-DD cells were treated with or without 200 nM Shield1 in the absence or in the presence of 100 nM Staurosporine. Cytochrome c, total and cleaved caspase 9 and caspase 3 levels in cytoplasmic “C” and mitochondrial “M” fractions were analyzed by Western blotting as described in the Material and Methods section. Tubulin was used to normalize cytoplasmic fractions and TOMM22 to normalize mitochondrial fractions. A representative blot of three independent experiments is shown.</p> <p>C) GH4C1-M-DD cells were treated with or without 200 nM Shield1 for 12 hours and then for 48h in the absence or in the presence of 100 nM Staurosporine. Bax, and BCl2 protein levels were analyzed by Western blot as described in the Material and Methods section. The figure shows a representative blot among three independent experiments. Ctrl= control. Str= Staurosporine.</p> <p>(D) GH4C1 were transfected with the pPTunerC Magmas- DD vector and then incubated in 96-well plates for 48 h in culture medium supplemented with or without 100 nM Staurosporine, in the absence or in the presence of 200 nm Shield1. The JC-1 mitochondrial membrane potential assay was preformed (see material and methods). The data are expressed as the ratio between red and green fluorescence ± SE in at least three independent experiments. *P<0.05.</p></div
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