ucn-1028-c and Glioblastoma

Calphostin C (cal-C) is a photoactivatable inhibitor that binds to the regulatory domain of protein kinase C (PKC) and to other proteins that contain diacylglycerol/phorbol ester binding sites. Cal-C is cytotoxic against many types of cancer cells, yet the basis for this activity remains poorly understood. Here, we show that one of the earliest effects of cal-C is an impairment of glycoprotein export from the endoplasmic reticulum (ER), accompanied by formation of ER-derived vacuoles. Vacuolization of the ER is correlated with induction of an ER stress response that includes activation of c-Jun N-terminal kinase and protein kinase R-like ER kinase, as well as increased expression of CCAAT/enhancer binding protein homologous transcription factor (CHOP; GADD153). These effects of cal-C are not mimicked by staurosporine, an inhibitor of PKC catalytic activity, indicating that ER stress is due to interaction of cal-C with targets other than PKC. In conjunction with the induction of ER stress, breast carcinoma cells undergo caspase-dependent cell death with early activation of caspases 9 and 7 and cleavage of poly(ADP-ribose)polymerase. Reduction of CHOP expression by short hairpin RNA decreases the sensitivity of the cells to cal-C, suggesting that induction of apoptosis by cal-C is related, at least in part, to ER stress triggered by disruption of ER morphology and transport function. Antineoplastic drugs that work by inducting ER stress have shown promise in preclinical and clinical trials. Thus, the present findings raise the possibility that cal-C may be useful for photodynamic therapy based on induction of ER stress in some forms of cancer.

Topics: Amyloid beta-Protein Precursor; Apoptosis; Blotting, Western; Breast Neoplasms; Cytoplasm; Endoplasmic Reticulum; Enzyme Inhibitors; Female; Glioblastoma; Golgi Apparatus; Humans; Immunoprecipitation; Light; Naphthalenes; Protease Nexins; Protein Kinase C; Protein Transport; Receptors, Cell Surface; Transcription Factor CHOP; Tumor Cells, Cultured; Vacuoles

Protein kinase C alpha (PKCalpha) has been implicated in tumor development with high levels of PKCalpha expression being associated with various malignancies including glioblastomas and tumors of the breast and prostate. To account for its upregulation in these cancers, studies have suggested that PKCalpha plays a role in promoting cell survival. Here we show by siRNA depletion in U87MG glioma cells that a critical threshold level of PKCalpha protein expression is essential for their growth in the presence of serum and for their survival following serum deprivation. Derivation of PKCalpha wt and KO mouse embryo fibroblast cell lines confirms a role for PKCalpha in protecting cells from apoptosis induced by serum deprivation. Notably, PKCalpha was found to mediate chemo-protection in these fibroblastic cell lines. In U87MG cells PKCalpha does not confer chemoprotection though this likely reflects growth arrest associated with its depletion. To determine the requirements for catalytic function, comparison was made between distinct classes of PKC inhibitors. In contrast to loss of PKCalpha protein, inhibition of PKC kinase activity in glioma cell lines does not significantly inhibit growth or survival. Conversely, inhibition with calphostin C, which targets the regulatory domain of PKC, potently inhibits proliferation and induces apoptosis. Evidence is presented that it is the fully phosphorylated, folded form of PKCalpha that confers this activity-independent behaviour. These results indicate an essential pro-proliferative and pro-survival role for PKCalpha in glioma but question the use of ATP competitive inhibitors as therapeutics, either alone, or in combination with chemotoxic agents.

Topics: Adenosine Triphosphate; Animals; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Fibroblasts; Glioblastoma; Humans; Indoles; Maleimides; Mice; Mice, Knockout; Naphthalenes; Protein Kinase C-alpha; Protein Kinase Inhibitors; Rats; RNA, Small Interfering

Changes in plasma membrane electrical potential evoke signals that regulate the expressions of various genes in the nervous system. However, the role of glycogen synthase kinase 3beta (GSK-3beta) in this process has not been elucidated. Thus, this study was performed to examine whether membrane depolarization can regulate the phosphorylation of GSK-3beta and to identify the molecular mechanisms involved in this regulation. The depolarization by treating with 100 mm KCl for 5 min resulted in the undulating phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells, in H19 -7/IGF-IR rat embryonic hippocampal cells, and in PC12 rat pheochromocytoma cells, but not in A172 human glioblastoma cells. Cellular beta-catenin contents showed a temporal pattern similar to that of the Ser-9 phosphorylation of GSK-3beta. Treatment with wortmannin or calphostin C or the expression of dominant negative Akt inhibited phosphorylation of GSK-3beta at Ser-9 following the KCl-induced depolarization of SH-SY5Y cells. Moreover, pretreatment with okadaic acid or cyclosporin A blocked the dephosphorylation of GSK-3beta at Ser-9 at 0, 15, and 30 min after KCl-induced depolarization, and the activity of protein phosphatases (PP) 2A and 2B increased at these times. Treatment with nifedipine or calcium-free medium inhibited GSK-3beta dephosphorylation following membrane depolarization, and the amounts of co-immunoprecipitated GSK-3beta and PP2A changed in parallel with GSK-3beta dephosphorylation. Our study demonstrated that KCl-induced depolarization caused undulating GSK-3beta phosphorylation/dephosphorylation, which was regulated for the most part by phosphatidylinositol 3-kinase and Akt (phosphorylation) and PP2A and PP2B (dephosphorylation), respectively.

Topics: Androstadienes; Animals; Calcineurin; Calcium; Cell Line; Cell Line, Tumor; Cell Membrane; Enzyme Inhibitors; Glioblastoma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Humans; Immunoblotting; Immunoprecipitation; Microscopy, Confocal; Naphthalenes; PC12 Cells; Phosphoprotein Phosphatases; Phosphorylation; Potassium Chloride; Rats; Serine; Threonine; Time Factors; Transfection; Wortmannin

Modulation of neurotrophic factors to protect neurons from damage is proposed as a novel mechanism for the action of antidepressants. However, the effect of antidepressants on modulation of glial cell line-derived neurotrophic factor (GDNF), which has potent and widespread effects, remains unknown. Here, we demonstrated that long-term use of antidepressant treatment significantly increased GDNF mRNA expression and GDNF release in time- and concentration-dependent manners in rat C6 glioblastoma cells. Amitriptyline treatment also increased GDNF mRNA expression in rat astrocytes. GDNF release continued for 24 h following withdrawal of amitriptyline. Furthermore, following treatment with antidepressants belonging to several different classes (amitriptyline, clomipramine, mianserin, fluoxetine and paroxetine) significantly increased GDNF release, but which did not occur after treatment with non-antidepressant psychotropic drugs (haloperidol, diazepam and diphenhydramine). Amitriptyline-induced GDNF release was inhibited by U0126 (10 microM), a mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H-89 (1 microM), a protein kinase A inhibitor, calphostin C (100 nM), a protein kinase C inhibitor and PD 169316 (10 microM), a p38 mitogen-activated protein kinase inhibitor. These results suggested that amitriptyline-induced GDNF synthesis and release occurred at the transcriptional level, and may be regulated by MEK/MAPK signalling. The enhanced and prolonged induction of GDNF by antidepressants could promote neuronal survival, and protect neurons from the damaging effects of stress. This may contribute to explain therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.

Topics: Amitriptyline; Animals; Antidepressive Agents; Astrocytes; Butadienes; Cell Line; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glial Cell Line-Derived Neurotrophic Factor; Glioblastoma; Imidazoles; Isoquinolines; Kinetics; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Naphthalenes; Nerve Growth Factors; Nerve Tissue Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Psychotropic Drugs; Rats; Sulfonamides

To examine whether protein kinase C (PKC) contributes to p53-dependent WAF1 induction after heat treatment, the effects of calphostin C (CAL), a specific inhibitor of PKC, on WAF1 induction were analyzed by PKC activity and gel mobility-shift assays and Western blot analysis in human glioblastoma cell lines. Heat-induced accumulation of WAF1 in A-172 cells carrying wild-type p53 (wtp53) was suppressed by CAL in a dose-dependent manner. In T98G cells carrying mutant p53 (mp53), no significant accumulation of WAF1 was observed after heat treatment and CAL exerted no significant effects on this response of T98G cells. In accordance with the accumulation of WAF1, heat-induced activation of the binding ability of p53 to p53 consensus sequence (p53 CON) was suppressed by CAL in A-172 cells but no DNA-binding activity was observed in the mp53 in T98G cells. PKC in A-172 cells was activated rapidly (within 5 min) after heat treatment in the membrane fraction but not in the cytosolic fraction. When the cell lines were treated with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), WAF1 was accumulated in A-172 cells in a dose-dependent manner but not in T98G cells. In addition, the cellular contents of WAF1 after heating did not increase in A-172 cells transformed with mp53. These results suggest that PKC contributes to heat-induced signal transduction leading to p53-dependent WAF1 induction in a way that PKC is involved in the specific DNA-binding activation of p53.

Topics: Amino Acid Sequence; Cell Extracts; Cell Line, Transformed; Cell Nucleus; Consensus Sequence; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA; Enzyme Activation; Enzyme Inhibitors; Glioblastoma; Hot Temperature; Humans; Mutation; Naphthalenes; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Suppressor Protein p53