bafilomycin-a1 and Neuroblastoma

The efficiency of cisplatin (CDDP) is significantly hindered by the development of resistance during the treatment course. To gain a detailed understanding of the molecular mechanisms underlying the development of cisplatin resistance, we comparatively analyzed established a CDDP-resistant neuroblastoma cell line (UKF-NB-4

Topics: Cell Line, Tumor; Cell Shape; Chloroquine; Cisplatin; Clone Cells; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Ontology; Gene Regulatory Networks; Humans; Lysosomes; Macrolides; Neuroblastoma; Transcriptome; Up-Regulation

Alzheimer's disease (AD) is a complex and progressive neurological disorder, and amyloid-β (Aβ) has been recognized as the major cause of AD. Inhibiting Aβ production and/or enhancing the clearance of Aβ to reduce its levels are still the effective therapeutic strategies pursued in anti-AD research. In previous studies, we have reported that selenomethionine (Se-Met), a major form of selenium in animals and humans with significant antioxidant capacity, can reduce both amyloid-β (Aβ) deposition and tau hyperphosphorylation in a triple transgenic mouse model of AD. In this study, a Se-Met treatment significantly decreased the Aβ levels in Neuron-2a/AβPPswe (N2asw) cells, and the anti-amyloid effect of Se-Met was attributed to its ability to inhibit Aβ generation by suppressing the activity of BACE1. Furthermore, both the LC3-II/LC3-I ratio and the number of LC3-positive puncta were significantly decreased in Se-Met-treated cells, suggesting that Se-Met also promoted Aβ clearance by modulating the autophagy pathway. Subsequently, Se-Met inhibited the initiation of autophagy through the AKT-mTOR-p70S6K signaling pathway and enhanced autophagic turnover by promoting autophagosome-lysosome fusion and autophagic clearance. Our results further highlight the potential therapeutic effects of Se-Met on AD.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Autophagy; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation; Humans; Immunosuppressive Agents; Lactosylceramides; Macrolides; Microscopy, Electron, Transmission; Neuroblastoma; Oncogene Protein v-akt; Selenomethionine; Signal Transduction; Sirolimus; Transfection

The aim of this study was to compare the ability of the drugs doxorubicin and Bafilomycin-A1(Baf-A1)to promote an immune reaction following the induction of cell death in a mouse neuroblastoma model. Neuro-2a cells were cultured in medium containing doxorubicin or Baf-A1. Bone marrow-derived dendritic cells(BM-DCs)were co-cultured with neuro-2A cells that were grown in doxorubicin- or Baf-A1-containing media, and phagocytosis of neuro-2a cells by the BN-DCs was evaluated. Additionally, dead neuro-2a cells were co-cultured with CD8a + lymphocytes and BM-DCs, and the proliferation of CD8a + cells was evaluated. Interferon-g(IFN-g)production was used as an indexof the immune response. Dead neuro-2a cells treated with doxorubicin were phagocytosed effectively compared to the cells treated with Baf-A1. However, phagocytosis of cells treated with Baf-A1 was promoted after stimulation with CpG oligodeoxynucleotide (CpG-ODN). When CD8a + cells were co-cultured with BM-DCs and doxorubicin-treated neuro-2a cells, CD8a + lymphocyte proliferation was observed. There was no statistical difference in IFN-g secretion between the doxorubicin-treated and Baf-A1-treated cells. However, after stimulation by CpG-ODN, IFN-g production was more effectively observed in the Baf-A1-treated cells. Induction of cell death by doxorubicin or Baf-A1 could possibly enhance antitumor immunity in patients receiving chemotherapy for neuroblastoma. Selection of anti-tumor agents and stimulation of BM-DCs with a toll-like receptor (TLR) agonist is considered important in promoting antitumor activity after chemotherapy.

Topics: Animals; Apoptosis; Cell Line, Tumor; Doxorubicin; Female; Immunotherapy; Interferon-alpha; Macrolides; Mice; Neuroblastoma

This study aimed to determine whether bafilomycin A1 (Baf-A1), a vacuolar H-ATPase inhibitor, could promote an immune response after the induction of apoptosis in mouse neuroblastoma cells. Mouse neuro-2a cells were cultured in a medium containing Baf-A1, and apoptosis was evaluated by flow cytometry. To examine the influence in the phagocytic cell, CD11b spleen cells or bone marrow-derived dendritic cells (BM-DCs) were cocultured with Baf-A1-treated neuro-2a. Interferon-γ (IFN-γ) production was used as an index of the immune response, and CDDP was used as the negative control. When CD8α cells were cocultured with CD11b cells and Baf-A1-treated neuro-2a cells in the presence of CpG-oligodeoxynucleotide (CpG-ODN) (a toll-like receptor 9 [TLR-9] agonist), CD8α lymphocyte proliferation and secretion of IFN-γ were observed. Phagocytosis of apoptotic cells by BM-DCs was maximal after simultaneous stimulation with CpG-ODN and lipopolysaccharide (LPS; a TLR-4 agonist). IFN-γ secretion was maximal when Baf-A1-treated neuro-2a cells and CD8α lymphocytes were cocultured with BM-DCs and stimulated with CpG-ODN. In contrast, IFN-γ production was not increased when the cells were cultured with LPS. When cells were stimulated with both CpG-ODN and LPS, promotion of IFN-γ production by CpG-ODN was suppressed. Induction of apoptosis by Baf-A1 could possibly enhance antitumor immunity in patients receiving chemotherapy for neuroblastoma. Stimulation of BM-DCs with a TLR-9 agonist could promote antitumor activity after Baf-A1 treatment.

Topics: Animals; Antigen-Presenting Cells; Apoptosis; Bone Marrow Cells; CD8-Positive T-Lymphocytes; Cell Proliferation; Coculture Techniques; Dendritic Cells; Enzyme Inhibitors; Female; Flow Cytometry; Humans; Immunity, Cellular; Interferon-gamma; Lipopolysaccharides; Lymphocyte Activation; Macrolides; Mice; Mice, Inbred A; Neuroblastoma; Oligodeoxyribonucleotides; Phagocytosis; Tumor Cells, Cultured

Amyloid precursor protein (APP) metabolism is central to the pathogenesis of Alzheimer disease. We showed recently that the amyloid intracellular domain (AICD), which is released by gamma-secretase cleavage of APP C-terminal fragments (CTFs), is strongly increased in cells treated with alkalizing drugs (Vingtdeux, V., Hamdane, M., Bégard, S., Loyens, A., Delacourte, A., Beauvillain, J.-C., Buée, L., Marambaud, P., and Sergeant, N. (2007) Neurobiol. Dis. 25, 686-696). Herein, we aimed to determine the cell compartment in which AICD accumulates. We show that APP-CTFs and AICD are present in multivesicular structures. Multivesicular bodies contain intraluminal vesicles (known as exosomes) when released in the extracellular space. We demonstrate that APP, APP-CTFs, and AICD are integrated and secreted within exosomes in differentiated neuroblastoma and primary neuronal culture cells. Together with recent data showing that amyloid-beta is also found in exosomes, our data show that multivesicular bodies are essential organelles for APP metabolism and that all APP metabolites can be secreted in the extracellular space.

Topics: Amyloid; Animals; Brain; Cell Line, Tumor; Endosomes; Enzyme Inhibitors; Humans; Macrolides; Models, Biological; Neuroblastoma; Neurons; Organelles; Protein Structure, Tertiary; Rats; Rats, Wistar

Amyloid-beta (Abeta) is the principal protein constituent of 'senile plaques' and is a suspected mediator in Alzheimer's disease (AD). Senile plaques also contain acetylcholinesterase (AChE; EC 3.1.1.7), which may have a role in promoting Alphabeta-toxicity. We have found that Alphabeta can affect AChE expression in a neuron-like line, the N1E.115 neuroblastoma cell. When 1 micro mAlphabeta 1-42 or 25-35 was added for 24 h to differentiating N1E.115 in culture, AChE activity increased 30-40% in adherent cells, and 100% or more in nonadherent cells. The changes in both tetrameric (G4) and monomeric (G1) AChE forms were comparable. Turnover studies indicated that the elevation of AChE activity reflected slowed AChE degradation rather than accelerated synthesis. With a similar time course, Alphabeta also increased the quantity of muscarinic receptors on the plasma membrane. Immunocytochemistry for a lysosomal membrane protein (LAMP-1) indicated no change in abundance or localization of lysosomes in treated cells. But decreased labeling by pH-sensitive fluorescent dye pointed to an impairment of lysosomal acidification. We consider that the alteration of AChE expression after Alphabeta-exposure could reflect lysosomal dysfunction, and might itself enhance Alphabeta-toxicity.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Animals; Anti-Bacterial Agents; Antigens, CD; Cell Adhesion; Enzyme Activation; Enzyme Inhibitors; Extracellular Space; Hydrogen-Ion Concentration; Immunohistochemistry; Intracellular Fluid; Lysosomal Membrane Proteins; Lysosomes; Macrolides; Mice; Neuroblastoma; Peptide Fragments; Protein Processing, Post-Translational; Receptors, Muscarinic; Tumor Cells, Cultured

Tamoxifen has been reported to have numerous physiological effects that are independent of the estrogen receptor, including sensitization of resistant tumor cells to many chemotherapeutic agents. Drug-resistant cells sequester weak base chemotherapeutics in acidic organelles away from their sites of action in the cytosol and nucleus. This work reports that tamoxifen causes redistribution of weak base chemotherapeutics from acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle acidification (e.g., monensin, bafilomycin A1) cause a similar redistribution. Measurement of cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the rate of vesicular transport though the recycling and secretory pathways. Organellar acidification is required for many cellular functions, and its disruption could account for many of the side effects of tamoxifen.

Topics: Anti-Bacterial Agents; Biological Transport; Boron Compounds; Breast Neoplasms; Cytoplasm; Doxorubicin; Drug Resistance, Neoplasm; Endosomes; Female; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Lysosomes; Macrolides; Monensin; Neuroblastoma; Receptors, Estrogen; Tamoxifen; Transferrin; Tumor Cells, Cultured

The aim of this work was to examine the effects of changes in external K+ concentration (Ko) around its physiological value, of various K+ channels blockers, including internal Cs+, of vacuolar H(+)-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K+ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (Ko = 5 mM) the membrane potential was -60 +/- 1 mV. It was unchanged when Ko was decreased to 1 mM and was depolarized by 4 +/- 1 mV when Ko was increased to 10 mM. (ii) Internal Cs+ depolarized the membrane by 21 +/- 3 mV. (iii) The internal application of the vacuolar H(+)-ATPase inhibitors N-ethylmaleimide (NEM), NO3- and bafilomycin A1 (BFA) depolarized the membrane by 15 +/- 2, 18 +/- 2 and 16 +/- 2 mV, respectively. (iv) When NEM or BFA were added to the internal medium containing Cs+, the membrane was depolarized by 45 +/- 1 and 42 +/- 2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K+ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K+ channels and an electrogenic vacuolar type H(+)-ATPase.

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Cesium; Ethylmaleimide; Glioma; Macrolides; Membrane Potentials; Mice; Neuroblastoma; Nitrites; Ouabain; Potassium; Potassium Channels; Proton Pumps; Rats; Time Factors; Tumor Cells, Cultured