bafilomycin-a1 and Brain-Neoplasms

Autophagy is considered to be another restorative focus for the treatment of brain tumors. Although several research have demonstrated that melatonin induces autophagy in colon cancer and hepatoma cells, there has not been any direct evidence of whether melatonin is capable of inducing autophagy in human glioma cells.. In the present research, we report that melatonin or its agonist, agomelatine, induced autophagy in A172 and U87-MG glioblastoma cells for a concentration-and time-dependent way, which was significantly attenuated by treatment with luzindole, a melatonin receptor antagonist. Furthermore, by suppressing autophagy at the late-stage with bafilomycin A1 and early stage with 3-MA, we found that the melatonin-induced autophagy was activated early, and the autophagic flux was complete. Melatonin treatment alone did not induce any apoptotic changes in the glioblastoma cells, as measured by flow cytometry. Western blot studies confirmed that melatonin alone prominently upregulated the levels of Beclin 1 and LC3 II, which was accompanied by an increase in the expression of Bcl-2, whereas it had no effect on the expression of Bax in the glioblastoma cells. Remarkably, co-treatment with 3-MA and melatonin significantly enhanced the apoptotic cell population in the glioblastoma cells, along with a prominent decrease in the expression of bcl-2 and increase in the Bax expression levels, which collectively indicated that the disruption of autophagy triggers the melatonin-induced apoptosis in glioblastoma cells.. These results provide information indicating that melatonin may act as a common upstream signal between autophagy and apoptosis, which may lead to the development of new therapeutic strategies for glioma.

Topics: Acetamides; Antineoplastic Agents; Apoptosis; Autophagy; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Glioblastoma; Humans; Macrolides; Melatonin; Receptors, Melatonin; Tryptamines

Ubiquitin-conjugating enzyme E2C (UBE2C) is characterized as a crucial molecule in cancer cell growth that plays an essential role in the development of gliomas, but the detailed mechanisms have not been fully elucidated. In this study, we found that Forkhead box transcription factor M1 (FoxM1) overexpression increased UBE2C expression, whereas FoxM1 suppression inhibited UBE2C expression in glioma cells. In addition, high FoxM1/UBE2C expression was significantly correlated with poor prognosis in glioma. We subsequently demonstrated that UBE2C was a direct transcriptional target of FoxM1, and site-directed mutations markedly down-regulated UBE2C promoter activity. Moreover, UBE2C siRNA (si-UBE2C) significantly induced glioma cell autophagy and increased both mCherry-LC3 punctate fluorescence and LC3B-II/LC3-I expression. Notably, the si-UBE2C-induced decrease in cell viability was markedly inhibited by the autophagy inhibitor bafilomycin A1. The silencing of UBE2C resulted in a distinct inhibition of the PI3K-Akt-mTOR pathway, which functions in the negative modulation of autophagy. Collectively, our findings provide clinical and molecular evidence that FoxM1 promotes glioma progression by enhancing UBE2C transcription and that the inhibition of UBE2C partially induces autophagic glioma cell death. Thus, targeting the FoxM1-UBE2C axis has therapeutic potential in the treatment of gliomas.

Topics: Adolescent; Adult; Apoptosis; Autophagy; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Computational Biology; Female; Forkhead Box Protein M1; Gene Knockdown Techniques; Gene Silencing; Glioma; Humans; Kaplan-Meier Estimate; Macrolides; Male; Neuroprotection; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin-Conjugating Enzymes

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Imatinib mesylate, a tyrosine kinase inhibitor, is effective in the therapy of tumors including leukemias but not as a monotherapy for malignant glioma. Recently, it is thought that the adequate modulation of autophagy can enhance efficacy of anticancer therapy. The outcome of autophagy manipulation, however, seems to depend on the autophagy initiator, the combined stimuli, the extent of cellular damage and the type of cells, and it is not yet fully understood how we should modulate autophagy to augment efficacy of each anticancer therapy. In this study, we examined the effect of imatinib with or without different types of autophagy inhibitors on human malignant glioma cells. Imatinib inhibited the viability of U87-MG and U373-MG cells in a dose dependent manner and caused nonapoptotic autophagic cell death. Suppression of imatinib-induced autophagy by 3-methyladenine or small interfering RNA against Atg5, which inhibit autophagy at an early stage, attenuated the imatinib-induced cytotoxicity. In contrast, inhibition of autophagy at a late stage by bafilomycin A1 or RTA 203 enhanced imatinib-induced cytotoxicity through the induction of apoptosis following mitochondrial disruption. Our findings suggest that therapeutic efficiency of imatinib for malignant glioma may be augmented by inhibition of autophagy at a late stage, and that appropriate modulation of autophagy may sensitize tumor cells to anticancer therapy.

Topics: Adenine; Antineoplastic Agents; Autophagy; Autophagy-Related Protein 5; Benzamides; Brain Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cisplatin; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; Imatinib Mesylate; Macrolides; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Paclitaxel; Piperazines; Pyrimidines

Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors tochemo- and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to gamma-radiation, has the capacity to repopulate the tumors and contribute to their malignant progression. gamma-radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by gamma-radiation in CD133+ GSCs. Irradiation of CD133+ cells induced autophagy within 24-48 hr and slightly decreased the viability of the cells. gamma-radiation induced a larger degree of autophagy in the CD133+ cells as compared with CD133- cells and the CD133+ cells expressed higher levels of the autophagy-related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD133+ cells to gamma-radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to gamma-radiation.

Topics: AC133 Antigen; Antigens, CD; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 12; Autophagy-Related Protein 5; Beclin-1; Brain Neoplasms; Electrochemotherapy; Gamma Rays; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioma; Glycoproteins; Humans; Macrolides; Membrane Proteins; Microtubule-Associated Proteins; Peptides; Radiation Tolerance; Radiation-Sensitizing Agents; Small Ubiquitin-Related Modifier Proteins; Up-Regulation

Autophagy is a novel response of cancer cells to ionizing radiation (IR) or chemotherapy, but its significance or mechanism remains largely elusive. Autophagy is characterized with the prominent formation of autophagic vacuoles in the cytoplasm. It is a protein degradation system that involves autophagic/lysosomal compartment. The process begins with sequestering a portion of the cytoplasm, forming the autophagosome. The autophagosome then fuses with the lysosome and lyses its contents. To study radiation-induced autophagy with specific molecules, we assessed changes in the expression of microtubule-associated protein light chain 3 (LC3) and its intracellular distribution after IR in comparison with starvation-induced autophagy. First, we showed that IR induced cell cycle arrest and autophagy, but not apoptosis, in human malignant glioma U373-MG cells. Type II LC3, that is specifically associated with the membrane of the autophagosome, increased after IR and amino acid starvation. Exogenous LC3 distributed on punctate structures, indicative of the formation of autophagosomes. Autophagy inhibitors, 3-methyladenine and bafilomycin A1, radiosensitized U373-MG cells. Furthermore, gammaH2AX foci, that show the extent of DNA double-strand breaks, were more pronounced and prolonged in the cells treated with IR and autophagy inhibitors than in those cells treated with IR only. Our results suggest that autophagy inhibitors may represent a new application of radiosensitization for malignant glioma cells.

Topics: Adenine; Apoptosis; Autophagy; Brain Neoplasms; Cell Cycle; DNA Damage; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Macrolides; Microtubule-Associated Proteins; Radiation Tolerance

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.

Topics: Apoptosis; Blotting, Western; Brain Neoplasms; Caspase 8; Caspases; Cathepsin B; Cell Line, Tumor; Cell Nucleus; Ceramides; Chromatography, High Pressure Liquid; Cycloheximide; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Glioma; Glutathione; Humans; Macrolides; Microscopy, Fluorescence; Mitochondria; Mitosis; Oligopeptides; Oncogene Proteins, Viral; Oxidation-Reduction; Oxidative Stress; Oxygen; Protein Synthesis Inhibitors; Reactive Oxygen Species; Recombinant Proteins; Repressor Proteins; Retroviridae; RNA, Small Interfering; Signal Transduction; Temperature; Time Factors; Transfection; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Although hyperthermia has been used as a treatment of malignant brain tumors, it is not yet clear what is the mechanism of the cell growth inhibition by heat shock, especially by the temperature which has clinically been applied to tumor-brain border-zone, 42-43 degrees C. Therefore, we evaluated the change of U251-MG and U87-MG human malignant glioma cells after 43 degrees C-heat shock comparing with that of 45 degrees C. First, we observed that cell growth was transiently inhibited after 43 degrees C-heat shock for 3 or 5 days, in U251-MG or U87-MG cells, respectively, which was followed by regrowth. During the period of transient growth inhibition, mild G2/M arrest was observed. However, apoptosis was observed in only 2.7% or 1.5%, of 43 degrees C-heated cells, in U251-MG or U87-MG cells, respectively. Instead, transmission electron micrography showed the formation of vacuoles, degeneration of mitochondria, and autophagosomes. Moreover, in the both cell lines, flow-cytometric analysis with acridine orange revealed the induction of acidic vesicle organelles, which was blocked by 3-methyladenine (3-MA), suggesting the involvement of autophagy. Furthermore, while 3-MA did not increase the anti-tumor effect of 43 degrees C-heat shock, bafilomycin A1, another autophagy inhibitor, did significantly enhance the effect in U251-MG cells. Taken together, mild heat shock (43 degrees C for 2 h) causes autophagy and mild G2/M arrest, but does not induce apparent apoptosis in U251-MG and U87-MG glioma cells. Inhibition of autophagy with bafilomycin A1 may increase the anti-tumor efficacy of mild heat shock against some malignant glioma cells.

Topics: Adenine; Apoptosis; Autophagy; Brain Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Enzyme Inhibitors; Glioma; Hot Temperature; Humans; Macrolides; Organelles; Thermodynamics