benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Glioblastoma

Neuromyelitis Optica (NMO) is an autoimmune inflammatory disease that affects the optic nerves and spinal cord. The autoantibody is generated against the abundant water channel protein of the brain, Aquaporin 4 (AQP4). Of the two isoforms of AQP4, the shorter one (M23) often exists as a supramolecular assembly known as an orthogonal array of particles (OAPs). There have been debates about the fate of these AQP4 clusters upon binding to the antibody, the exact mechanism of its turnover, and the proteins associated with the process. Recently several clinical cases of NMO were reported delineating the effect of Rituximab (RTX) therapy. Extending these reports at the cell signaling level, we developed a glioma based cellular model that mimicked antibody binding and helped us track the subsequent events including a variation of AQP4 levels, alterations in cellular morphology, and the changes in downstream signaling cascades. Our results revealed the extent of perturbations in the signaling pathways related to stress involving ERK, JNK, and AKT1 together with markers for cell death. We could also decipher the possible routes of degradation of AQP4, post-exposure to antibody. We further investigated the effect of autoantibody on AQP4 transcriptional level and involvement of FOXO3a and miRNA-145 in the regulation of transcription. This study highlights the differential outcome at the cellular level when treated with the serum of the same patient pre and post RTX therapy and for the first time mechanistically describes the effect of RTX.

Topics: Adult; Aquaporin 4; Autoantibodies; Autoantigens; Cell Line, Tumor; Cell Membrane; Cell Shape; Cell Surface Extensions; Female; Forkhead Box Protein O3; Glioblastoma; Humans; Immunoglobulin G; Leupeptins; Male; MicroRNAs; Microscopy, Confocal; Neuromyelitis Optica; Proteasome Endopeptidase Complex; Rituximab; Signal Transduction; Single-Cell Analysis; Transcription, Genetic; Young Adult

Topics: Animals; Antineoplastic Agents; Apoptosis; Astrocytes; Caspases; Cell Line, Tumor; Cell Survival; Cisplatin; Etoposide; Glioblastoma; Humans; Leupeptins; Mice; Resveratrol; Tumor Suppressor Protein p53

Cell migration and invasion are highly regulated processes involved in both physiological and pathological conditions. Here we show that autophagy modulation regulates the migration and invasion capabilities of glioblastoma (GBM) cells. We observed that during autophagy occurrence, obtained by nutrient deprivation or by pharmacological inhibition of the mTOR complexes, GBM migration and chemokine-mediated invasion were both impaired. We also observed that SNAIL and SLUG, two master regulators of the epithelial-mesenchymal transition (EMT process), were down-regulated upon autophagy stimulation and, as a consequence, we found a transcriptional and translational up-regulation of N- and R-cadherins. Conversely, in BECLIN 1-silenced GBM cells, an increased migration capability and an up-regulation of SNAIL and SLUG was observed, with a resulting decrease in N- and R-cadherin mRNAs. ATG5 and ATG7 down-regulation also resulted in an increased migration and invasion of GBM cells combined to an up-regulation of the two EMT regulators. Finally, experiments performed in primary GBM cells from patients largely confirmed the results obtained in established cell cultures. Overall, our results indicate that autophagy modulation triggers a molecular switch from a mesenchymal phenotype to an epithelial-like one in GBM cellular models. Since the aggressiveness and lethality of GBM is defined by local invasion and resistance to chemotherapy, we believe that our evidence provides a further rationale for including autophagy/mTOR-based targets in the current therapeutical regimen of GBM patients.

Topics: Animals; Autophagy; Cell Line, Tumor; Cell Movement; Chloroquine; Culture Media, Serum-Free; Epithelial-Mesenchymal Transition; Glioblastoma; Humans; Leupeptins; Mice; Naphthyridines; Neoplasm Invasiveness; Up-Regulation

The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is regulated by the p53 and Egr-1 tumor suppressor pathways. Many anti-cancer drugs and chemicals induce NAG-1 expression, but the mechanisms are not fully understood. Transgenic mice expressing human NAG-1 are resistant to intestinal and prostate cancer, suggesting that NAG-1 is a tumor suppressor. Proteasome inhibitors exhibit anti-glioblastoma activities in preclinical studies. Here, we show that the proteasome inhibitors MG132 and bortezomib induced NAG-1 expression and secretion in glioblastoma cells. MG132 increased NAG-1 expression through transcriptional and post-transcriptional mechanisms. At the transcriptional level, the induction of NAG-1 required the -133 to +41 bp region of the promoter. At post-transcriptional levels, MG132 stabilized NAG-1 mRNA by increasing the half-life from 1.5 h to >8 h. Because of the dramatic increase in mRNA stability, this is likely the major contributor to MG132-mediated NAG-1 induction. Further probing into the mechanism revealed that MG132 increased phosphorylation of the p38 MAPK pathway. Consequently, inhibiting p38 phosphorylation blocked activation of the NAG-1 promoter and decreased mRNA stability, indicating that p38 MAPK activation mediates both MG132-dependent promoter activation and mRNA stabilization of NAG-1. We propose that the induction of NAG-1 by p38 MAPK is a potential contributor to the anti-glioblastoma activity of proteasome inhibitors.

Topics: Animals; Brain Neoplasms; Cysteine Proteinase Inhibitors; Glioblastoma; Growth Differentiation Factor 15; Humans; Leupeptins; Mice; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Proteasome Inhibitors; RNA Stability; RNA, Messenger

Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21(WAF1) protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.

Topics: Adjuvants, Pharmaceutic; Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Caspase 3; Cell Cycle; Cell Line, Tumor; Cisplatin; Doxorubicin; Glioblastoma; Humans; Leupeptins; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinases; NF-kappa B; Paclitaxel; Phosphoinositide-3 Kinase Inhibitors; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Astrocytes; Brain Neoplasms; Cell Cycle; Cell Death; Cell Line, Tumor; Cisplatin; Curcumin; Doxorubicin; Drug Synergism; Glioblastoma; Humans; Leupeptins; Molecular Targeted Therapy; NF-kappa B; Nitriles; Oxides; Rats; Sesquiterpenes; Signal Transduction; Sulfones

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

Topics: Active Transport, Cell Nucleus; Apoptosis; Cell Nucleus; DNA-Binding Proteins; Doxorubicin; Drug Resistance, Neoplasm; Glioblastoma; Humans; Leupeptins; NF-kappa B; Proteasome Inhibitors; TNF-Related Apoptosis-Inducing Ligand; Transcriptional Activation; Tumor Cells, Cultured

The ellipticinium and its derivatives have been studied as anti-cancer agents with preferentially cyto-toxicity to the brain tumor cell lines. During the course of our study to determine whether an ellipticine derivative, API59-Cl would sensitize radio-resistant U87 glioblastoma cells to radiation, we found that it reduced the level of p27, a cyclin-dependent kinase inhibitor. API59-Cl induced a dose and time dependent p27 reduction in U87 cells. The compound-induced p27 reduction was also seen in three additional glioblastoma lines, T98G, U251 and U118 as well as in mouse embryonic fibroblasts. Mechanistic study of API59-Cl mediated p27 reduction revealed that it was not due to an altered p27 transcription, rather due to a shortened protein half-life as a result of enhanced p27 degradation. Indeed, API59-Cl induced p27 degradation was dependent on ubiquitin-proteasome pathway, particularly E3 ubiquitin ligase component, Skp2, but not Cullin-4A/4B, and can be largely blocked by proteasome inhibitors MG132 or PS341. Finally, we demonstrated that API59-Cl inhibited U87 cell growth with an IC50 of 1.7 muM, which is independent of its p27 degrading activity. This is the first report, to our knowledge, that the ellipticinium class of small molecule compounds promotes p27 degradation via ubiquitin-proteasome pathway. The finding could provide a new tool to further understand the mechanism of p27 degradation.

Topics: Animals; Antineoplastic Agents, Phytogenic; Boronic Acids; Bortezomib; Brain Neoplasms; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Proteinase Inhibitors; Ellipticines; Glioblastoma; Humans; Leupeptins; Mice; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Radiation-Sensitizing Agents; S-Phase Kinase-Associated Proteins; Transcription, Genetic; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases

In contrast to nuclear factor-kappaB (NF-kappaB) activation by tumor necrosis factor-alpha (TNF-alpha), the specific processes involved in the activation of this transcription factor by ionizing radiation (IR) have not been completely defined. According to the classical paradigm, a critical event in NF-kappaB activation is the degradation of I(kappa)B(alpha). Data presented herein show that, in contrast to treatment with TNF-alpha, IR-induced NF-kappaB activation was not accompanied by degradation of I(kappa)B(alpha) in the U251 glioblastoma cell line as determined in whole cell lysates. However, treatment with the proteosome inhibitor MG-132 inhibited NF-kappaB activation induced by IR, suggesting that I(kappa)B(alpha) degradation was a critical event in this process. To reconcile these results, U251 cell lysates were separated into soluble and insoluble fractions and I(kappa)B(alpha) levels evaluated. Although I(kappa)B(alpha) was found in both subcellular fractions, treatment with IR resulted in the degradation of I(kappa)B(alpha) only in the insoluble fraction. Further subcellular fractionation suggested that the IR-sensitive, insoluble pool of I(kappa)B(alpha) was associated with the plasma membrane. These data suggest that the subcellular location of I(kappa)B(alpha) is a critical determinant in IR-induced NF-kappaB activation.

Topics: Cell Fractionation; Cell Membrane; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Enzyme Activation; Glioblastoma; Humans; I-kappa B Proteins; Leupeptins; NF-kappa B; NF-KappaB Inhibitor alpha; Radiation, Ionizing; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha