benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Medulloblastoma

Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-Associated Death Protein; Boronic Acids; Bortezomib; Caspase 3; Caspase 9; Cerebellar Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Humans; Medulloblastoma; NF-kappa B; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazines; Sorafenib; Tumor Cells, Cultured

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytochrome c Group; Death-Associated Protein Kinases; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Medulloblastoma; Microscopy, Fluorescence; Mitochondria; Mutation; Neuroblastoma; Oligopeptides; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured

Chemotherapeutic agents and gamma-irradiation used in the treatment of brain tumors, the most common solid tumors of childhood, have been shown to act primarily by inducing apoptosis. Here, we report that activation of the CD95 pathway was involved in drug- and gamma-irradiation-induced apoptosis of medulloblastoma and glioblastoma cells. Upon treatment CD95 ligand (CD95-L) was induced that stimulated the CD95 pathway by crosslinking CD95 via an autocrine/paracrine loop. Blocking CD95-L/receptor interaction using F(ab')2 anti-CD95 antibody fragments strongly reduced apoptosis. Apoptosis depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3 like proteases) as it was almost completely abrograted by the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Apoptosis was mediated by cleavage of the receptor proximal caspase FLICE/MACH (caspase-8) and the downstream caspase CPP32 (caspase-3, Apopain) resulting in cleavage of the prototype caspase substrate PARP. Moreover, CD95 was upregulated in wild-type p53 cells thereby increasing responsiveness towards CD95 triggering. Since activation of the CD95 system upon treatment was also found in primary medulloblastoma cells ex vivo, these findings may have implications to define chemosensitivity and to develop novel therapeutic strategies in the management of malignant brain tumors.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Brain Neoplasms; Caspase 3; Caspase 8; Caspase 9; Caspases; Cerebellar Neoplasms; Cesium Radioisotopes; Cisplatin; Cycloheximide; Doxorubicin; fas Receptor; Flow Cytometry; Gamma Rays; Glioblastoma; Humans; Immunoglobulin Fab Fragments; Medulloblastoma; Neuroprotective Agents; Tumor Cells, Cultured