cytochrome-c-t and Glioblastoma

Glioma is the foremost recurrent type of brain tumor in humans; in particular, glioblastoma (GBM) is the main form of brain tumor (GBM) that is highly proliferative and impervious to apoptosis. Triphlorethol-A (TA), a phlorotannin isolated from Ecklonia cava, exhibited cytoprotective, antioxidant, and anticancer properties. However, the exact molecular action of TA in the U251 human GBM cells remains unknown. This may be the first report on the antiproliferative and apoptotic mechanisms of TA on GBM. The cytotoxicity, intracellular reactive oxygen species (ROS), matrix metalloproteinase (MMP), and cell apoptosis activity of TA have been evaluated by the MTT assay and by DCFH-DA, Rh-123, AO/EB, and western blot analysis. The results obtained showed that TA abridged the viability of U251 cells, while MMP increased apoptosis by increasing the ROS levels in a time-dependent manner. The results showed that a reduction in U251 cell proliferation was associated with the regulation of JAK2/STAT3 and p38 MAPK/ERK signaling pathways. TA was found to suppress pJAK, pSTAT3, p38 MAPK, and pERK phosphorylation, thereby causing Bax/Bcl-2 imbalance, activating the caspase cascade and cytochrome c, and inducing apoptosis. Our findings showed that the suppression of JAK2/STAT3 and p38 MAPK/ERK signaling by TA results in cell growth arrest and stimulation of apoptosis in GBM cells. These studies justify the protective remedy of TA against GBM.

Topics: Antioxidants; Apoptosis; bcl-2-Associated X Protein; Brain Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Glioblastoma; Glioma; Humans; Janus Kinase 2; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phloroglucinol; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; STAT3 Transcription Factor

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Temozolomide (TMZ) is the first-line chemotherapeutic drug for treating GBM. However, drug resistance is still a challenging issue in GBM therapy. Our preliminary results showed upregulation of

Topics: Apoptosis; bcl-2-Associated X Protein; Benzamides; Brain Neoplasms; Caspase 6; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Glioblastoma; Humans; Male; Mitochondria; Nitriles; Phenylthiohydantoin; Receptors, Androgen; RNA; RNA, Messenger; Temozolomide

The cross-talk between the mitochondrion and the nucleus regulates cellular functions, including differentiation and adaptation to stress. Mitochondria supply metabolites for epigenetic modifications and other nuclear-associated activities and certain mitochondrial proteins were found in the nucleus. The voltage-dependent anion channel 1 (VDAC1), localized at the outer mitochondrial membrane (OMM) is a central protein in controlling energy production, cell growth, Ca

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Neoplasms; Caspases; Cell Differentiation; Cell Nucleus; Cytochromes c; Epigenesis, Genetic; Glioblastoma; Histones; Humans; Lymphoma, Large B-Cell, Diffuse; Male; Mice, Nude; Mitochondrial Proteins; Receptors, GABA; Tumor Suppressor Protein p53; Voltage-Dependent Anion Channel 1; Xenograft Model Antitumor Assays

Apoptosis induction of cancer cells can be an appropriate strategy by which chemotherapeutic agents kill tumor cells. The aim of the present study was to investigate the effect of temozolomide and thymoquinone combination on apoptotic pathway of human glioblastoma multiforme cell line (U87MG). U87MG cells were cultured, treated with temozolomide and thymoquinone, and cell proliferation was measured. Apoptosis cell death and its possible mechanism were investigated by various methods. Combination of temozolomide and thymoquinone had a synergistic effect on cells viability. Thymoquinone intensified the temozolomide-induced apoptosis. Combination of temozolomide and thymoquinone can be a good strategy for treatment of glioblastoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzoquinones; Brain Neoplasms; Cell Line, Tumor; Comet Assay; Cytochromes c; Dacarbazine; Drug Synergism; Glioblastoma; Glutathione; Humans; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Nitric Oxide; Reactive Oxygen Species; Staining and Labeling; Temozolomide

Glioblastoma multiforme (GBM), a primary brain malignancy characterized by high morbidity, invasiveness, proliferation, relapse and mortality, is resistant to chemo- and radiotherapies and lacks effective treatment. GBM tumors undergo metabolic reprograming and develop anti-apoptotic defenses. We targeted GBM with a peptide derived from the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), a key component of cell energy, metabolism and apoptosis regulation. VDAC1-based cell-penetrating peptides perturbed cell energy and metabolic homeostasis and induced apoptosis in several GBM and GBM-derived stem cell lines. We found that the peptides simultaneously attacked several oncogenic properties of human U-87MG cells introduced into sub-cutaneous xenograft mouse model, inhibiting tumor growth, invasion, and cellular metabolism, stemness and inducing apoptosis. Peptide-treated tumors showed decreased expression of all tested metabolism-related enzymes and transporters, and elevated levels of apoptotic proteins, such as p53, cytochrome c and caspases. Retro-Tf-D-LP4, containing the human transferrin receptor (TfR)-recognition sequence, crossed the blood-brain barrier (BBB) via the TfR that is highly expressed in the BBB to strongly inhibit tumor growth in an intracranial xenograft mouse model. In summary, the VDAC1-based peptides tested here offer a potentially affordable and innovative new conceptual therapeutic paradigm that might overcome GBM stemness and invasiveness and reduce relapse rates.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Survival; Cell-Penetrating Peptides; Cytochromes c; Disease Models, Animal; Gene Expression; Glioblastoma; Hexokinase; Humans; Membrane Potential, Mitochondrial; Mice; Mitochondria; Models, Biological; Neoplastic Stem Cells; Peptides; Voltage-Dependent Anion Channel 1

Backgroud:Glioblastoma is a kind of highly malignant and aggressive tumours in the central nervous system. Previously, we found that neurotensin (NTS) and its high-affinity receptor 1 (NTSR1) had essential roles in cell proliferation and invasiveness of glioblastoma. Unexpectedly, cell death also appeared by inhibition of NTSR1 except for cell cycle arrest. However, the mechanisms were remained to be further explored.. Cells treated with SR48692, a selective antagonist of NTSR1, or NTSR1 shRNA were stained with Annexin V-FITC/PI and the apoptosis was assessed by flow cytometry. Cytochrome c release was detected by using immunofluorescence. Mitochondrial membrane potential (MMP, ΔΨm) loss was stained by JC-1 and detected by immunofluorescence or flow cytometry. Apoptosis antibody array and microRNA microarray were performed to seek the potential regulators of NTSR1 inhibition-induced apoptosis. Interaction between let-7a-3p and Bcl-w 3'UTR was evaluated by using luciferase assay.. SR48692 induced massive apoptosis, which was related to mitochondrial cytochrome c release and MMP loss. Knockdown of NTSR1 induced slight apoptosis and significant MMP loss. In addition, NTSR1 inhibition sensitised glioblastoma cells to actinomycin D or doxorubicin-induced apoptosis. Consistently, NTSR1 inhibition-induced mitochondrial apoptosis was accompanied by downregulation of Bcl-w and Bcl-2. Restoration of Bcl-w partly rescued NTSR1 deficiency-induced apoptosis. In addition, NTSR1 deficiency promoted higher let-7a-3p expression and inhibition let-7a-3p partly rescued NTSR1 inhibition-induced apoptosis. In addition, let-7a-3p inhibition promoted 3'UTR activities of Bcl-w and the expression of c-Myc and LIN28, which were the upstream of let-7a-3p, decreased after NTSR1 inhibition.. NTSR1 had an important role in protecting glioblastoma from intrinsic apoptosis via c-Myc/LIN28/let-7a-3p/Bcl-w axis.

Topics: 3' Untranslated Regions; Animals; Antibiotics, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cell Line, Tumor; Cytochromes c; Dactinomycin; Down-Regulation; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Glioblastoma; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; MicroRNAs; Mitochondria; Neoplasm Transplantation; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Quinolines; Receptors, Neurotensin; RNA, Small Interfering; Up-Regulation

Proteins initially identified as essential for apoptosis also mediate a wide range of non-apoptotic functions that include cell cycle progression, differentiation and metabolism. As this phenomenon was mostly reported with non-cancer cells, we considered non-conventional roles for the apoptotic machinery in the cancer setting. We found that treating glioblastoma (GBM) tumors with siRNA against VDAC1, a mitochondrial protein found at the crossroads of metabolic and survival pathways and involved in apoptosis, inhibited tumor growth while leading to differentiation of tumor cells into neuronal-like cells, as reflected in the expression of specific markers. Although VDAC1 depletion did not induce apoptosis, the expression levels of several pro-apoptotic regulatory proteins were changed. Specifically, VDAC1 deletion led to up-regulation of caspases, p53, cytochrome c, and down-regulation of SMAC/Diablo, AIF and TSPO. The down-regulated group was highly expressed in U-87MG xenografts, as well as in GBMs from human patients. We also showed that the rewired cancer-cell metabolism resulting from VDAC1 depletion reinforced cell growth arrest and differentiation via alterations in the transcription factors p53, c-Myc, HIF-1α and NF-κB. The decrease in c-Myc, HIF-1α and NF-κB levels was in accord with reduced cell proliferation, whereas increased p53 expression promoted differentiation. Thus, upon metabolic re-programing induced by VDAC1 depletion, the levels of pro-apoptotic proteins associated with cell growth decreased, while those connected to cell differentiation increased, converting GBM cells into astrocyte- and neuron-like cells. The results reveal that in tumors, pro-apoptotic proteins can perform non-apoptotic functions, acting as regulators of cell growth and differentiation, making these molecules potential new targets for cancer therapy. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Brain Neoplasms; Caspases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Mice; Mice, Nude; Mitochondria; Mitochondrial Proteins; Neurons; NF-kappa B; Proto-Oncogene Proteins c-myc; Receptors, GABA; RNA, Small Interfering; Signal Transduction; Tumor Suppressor Protein p53; Voltage-Dependent Anion Channel 1; Xenograft Model Antitumor Assays

Identification of therapeutic strategies that might enhance the efficacy of B-cell lymphoma-2 (Bcl-2) inhibitor ABT-737 [N-{4-[4-(4-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide] is of great interest in many cancers, including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt are currently being assessed clinically in patients with glioma. Because PI3K/Akt inhibition would be expected to have many proapoptotic effects, we hypothesized that there may be unique synergy between PI3K inhibitors and Bcl-2 homology 3 mimetics. Toward this end, we assessed the combination of the PI3K/Akt inhibitor NVP-BKM120 [5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine] and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8 and -3, PARP, and cell death, irrespective of PTEN status. The enhanced lethality observed with this combination also appears dependent on the loss of mitochondrial membrane potential and release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, and activation of Bax and Bak caused by these inhibitors were the key factors for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony-forming ability, suggesting the potential use of NVP-BKM120 as a promising agent to improve the anticancer activities of ABT-737.

Topics: Aminopyridines; Antineoplastic Agents; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Biphenyl Compounds; Caspase 3; Caspase 8; Caspases; Cell Death; Cell Line, Tumor; Chromosome Segregation; Cytochromes c; DNA Damage; Drug Synergism; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Morpholines; Nitrophenols; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Poly(ADP-ribose) Polymerases; Primary Cell Culture; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides

Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched.. The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded.. All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident.. This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Survival; Central Nervous System Neoplasms; Chromans; Cytochromes c; DNA Fragmentation; Glioblastoma; Humans; Isomerism; Lung Neoplasms; Mitochondria; Tocotrienols; Vitamin E

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.

Topics: Apoptosis; Azoles; bcl-2-Associated X Protein; Buthionine Sulfoximine; Cell Line, Tumor; Cell Survival; Coculture Techniques; Cytochromes c; Cytosol; Ethacrynic Acid; Glioblastoma; Glutathione; Humans; Isoindoles; Mitochondria; Neuroblastoma; Neurons; Organoselenium Compounds; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Glioblastoma multiforme is the most aggressive primary brain tumour. At the cellular and molecular levels, several mechanisms responsible for apoptosis or autophagy induction are blocked. Identification of molecular targets stimulating cells to initiate programmed cell death should be performed for therapeutic purposes. A promising solution is the combination of temozolomide and quercetin. The aim of our study was to evaluate the effect of both drugs, applied alone and in combinations, on apoptosis and autophagy induction in human glioblastoma multiforme T98G cells. Our results clearly indicate that quercetin and temozolomide induce apoptosis very significantly, having no effect on autophagy induction. At the molecular level, it was correlated with caspase 3 and 9 activation, cytochrome c release from the mitochondrium and a decrease in the mitochondrial membrane potential. Both drugs are also potent Hsp27 and Hsp72 inhibitors. This suggests that the apoptotic signal goes through an internal pathway. Increased expression of caspase 12 and the presence of several granules in the cytoplasm after temozolomide treatment with or without quercetin preceding appearance of apoptosis may suggest that apoptosis is initiated by ER stress. Additionally, it was accompanied by changes in the nuclear morphology from circular to 'croissant like'.

Topics: Antineoplastic Agents, Alkylating; Antioxidants; Apoptosis; Brain Neoplasms; Caspase 12; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Dacarbazine; Endoplasmic Reticulum Stress; Enzyme Activation; Glioblastoma; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria; Molecular Chaperones; Quercetin; Temozolomide

The aim of the present study was to investigate whether silencing of Hsp27 or Hsp72 expression in glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cells increases their sensitivity to programmed cell death induction upon temozolomide and/or quercetin treatment. Transfection with specific siRNA was performed for the Hsp gene silencing. As revealed by microscopic observation and flow cytometry, the inhibition of Hsp expression was correlated with severe apoptosis induction upon the drug treatment studied. No signs of autophagy were detected. This was correlated with a decreased mitochondrial membrane potential, increased level of cytochrome c in the cytoplasm, and activation of caspase 3 and caspase 9. All these results suggest that the apoptotic signal was mediated by an internal pathway. Additionally, in a large percentage of cells treated with temozolomide, with or without quercetin, granules within the ER system were found, which was accompanied by an increased level of caspase 12 expression. This might be correlated with ER stress. Quercetin and temozolomide also changed the shape of nuclei from circular to "croissant like" in both transfected cell lines. Our results indicate that blocking of Hsp27 and Hsp72 expression makes T98G cells and MOGGCCM cells extremely vulnerable to apoptosis induction upon temozolomide and quercetin treatment and that programmed cell death is initiated by an internal signal.

Topics: Apoptosis; Astrocytoma; Autophagy; Caspase 12; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Dacarbazine; Endoplasmic Reticulum Stress; Gene Silencing; Glioblastoma; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Membrane Potential, Mitochondrial; Molecular Chaperones; Quercetin; Temozolomide

Glioblastomas are invasive tumors with poor prognosis despite current therapies. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth, and we and others have noted some antiglioma activity from HDACIs, such as vorinostat, although insufficient to warrant use as monotherapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with vorinostat treatment alone, the combination of vorinostat plus bortezomib significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib with vorinostat enhanced apoptosis by increasing Mcl-1 cleavage, Noxa upregulation, Bak and Bax activation, and cytochrome c release. Further downregulation of Mcl-1 using shRNA enhanced cell killing by the bortezomib/vorinostat combination. Vorinostat induced a rapid and sustained phosphorylation of histone H2AX in primary GBM and T98G cells, and this effect was significantly enhanced by co-administration of bortezomib. Vorinostat/bortezomib combination also induced Rad51 downregulation, which plays an important role in the synergistic enhancement of DNA damage and apoptosis. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Boronic Acids; Bortezomib; Cell Line, Tumor; Central Nervous System Neoplasms; Cytochromes c; DNA Damage; Glioblastoma; Glioma; Histones; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Phosphorylation; Proteasome Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Reactive Oxygen Species; Tumor Cells, Cultured; Vorinostat

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mRNAs. miR-125b, one of the neuronal miRNAs, was recently found to be necessary for stem cell fission and for making stem cells insensitive to chemotherapy signals. Temozolomide (TMZ) is a promising chemotherapeutic agent for treating glioblastomas. However, resistance develops quickly and with a high frequency. Given the insensitivity of some glioblastomas to TMZ and the hypothesis that glioma stem cells cause resistance to drug therapy, exploring the functions and mechanisms of miR-125b action on TMZ-treated glioblastoma stem cells would be valuable. In this study, we found that miR-125b-2 is overexpressed in glioblastoma multiforme tissues and the corresponding stem cells (GBMSC); downregulation of miR-125b-2 expression in GBMSC could allow TMZ to induce GBMSC apoptosis. Additionally, the expression of the anti-apoptotic protein Bcl-2 was decreased after the TMZ+miR-125b-2 inhibitor treatment, while the expression of the proapoptotic protein Bax was increased. Further research demonstrated that the induction of apoptosis in GBMSC is also associated with increased cytochrome c release from mitochondria, induction of Apaf-1, activation of caspase-3 and poly-ADP-ribose polymerase (PARP). Taken together, these results suggest that miR-125b-2 overexpression might confer glioblastoma stem cells resistance to TMZ.

Topics: Adult; Antineoplastic Agents, Alkylating; Apoptosis; Cytochromes c; Dacarbazine; Drug Resistance, Neoplasm; Glioblastoma; Humans; MicroRNAs; Mitochondria; Neoplastic Stem Cells; Temozolomide; Tumor Cells, Cultured

Novaeguinoside II is one of the newly found asterosaponins, which are a kind of steroidal glycosides extracted from starfish with an anti-tumor effect. In this study, we investigated the bioactivity of novaeguinoside II on human U87MG glioblastoma cells. The results showed that novaeguinoside II significantly suppresses U87MG cell proliferation in a time- and concentration-dependent manner. Flow cytometric analysis of DNA in U87MG cells showed that novaeguinoside II induces the prominent appearance of an S phase peak in the cell cycle that is suggestive of apoptosis that is identical to the result of an annexin V/PI assay. Fluorescence and electron microscopy revealed apoptotic change of U87MG cells. The electrophoresis of DNA showed a typical "ladder" that is consistent with apoptotic DNA fragmentation. Cytofluorometry showed a decreased mitochondrial transmembrane potential in novaeguinoside II-treated U87MG cells. Western blot showed that novaeguinoside II increased the expression of cytochrome-c and caspase-3 protein. The novel findings suggest that novaeguinoside II can induce apoptosis of U87MG cells by a mitochondrial apoptotic pathway.

Topics: Annexin A5; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Glioblastoma; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Saponins; Time Factors

Apoptosis is a highly conserved genetic process leading to death in mammalian cells. A critical step in apoptosis is mitochondrial membrane permeabilization, which results in the release of proteins critical to downstream events. Transmembrane protein 14A (TMEM14A) was identified as a novel suppressor of Bax using yeast-based functional screening. TMEM14A is a novel mitochondria-associated membrane protein containing a putative transmembrane domain. Over-expression of TMEM14A in U87MG cells inhibited N-(4-hydroxyphenyl)retinamide (4-HPR)-induced apoptosis. TMEM14A prevented 4-HPR-induced loss of mitochondrial membrane potential (MMP), the release of cytochrome c, and the activation of caspase-3, but not the generation of reactive oxygen species, suggesting that TMEM14A regulates mitochondrial membrane potential in a ROS-independent manner. As expected, cyclosporin A, an inhibitor of membrane potential transition, inhibited 4-HPR-induced loss of MMP and apoptosis in U87MG cells, indicating that loss of MMP plays a pivotal role in 4-HPR-induced apoptosis. Suppression of TMEM14A expression using shRNA significantly increased apoptosis and MMP loss in untreated and 4-HPR-treated cells. These findings show for the first time that TMEM14A inhibits apoptosis by blocking the mitochondrial permeability transition and stabilizing mitochondrial membrane potential.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cyclosporine; Cytochromes c; Enzyme Inhibitors; Fenretinide; Flow Cytometry; Glioblastoma; Humans; Immunoblotting; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering

Glioblastoma shows poor response to current therapies and warrants new therapeutic strategies. We examined the efficacy of combination of valproic acid (VPA) and taxol (TX) or nanotaxol (NTX) in human glioblastoma LN18 and T98G cell lines. Cell differentiation was manifested in changes in morphological features and biochemical markers. Cell growth was controlled with down regulation of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), nuclear factor-kappa B (NF-κB), phospho-Akt (p-Akt), and multi-drug resistance (MDR) marker, indicating suppression of angiogenic, survival, and multi-drug resistance pathways. Cell cycle analysis showed that combination therapy (VPA and TX or NTX) increased the apoptotic sub G1 population and apoptosis was further confirmed by Annexin V-FITC/PI binding assay and scanning electron microscopy. Combination therapy caused activation of caspase-8 and cleavage of Bid to tBid and increased Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Upregulation of calpain and caspases (caspase-9 and caspase-3) and substrate degradation were also detected in course of apoptosis. The combination of VPA and NTX most effectively controlled the growth of LN18 and T98G cells. Therefore, this combination of drugs can be used as an effective treatment for controlling growth of human glioblastoma cells.

Topics: Albumin-Bound Paclitaxel; Albumins; Apoptosis; Apoptosis Inducing Factor; ATP Binding Cassette Transporter, Subfamily B, Member 1; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 8; Caspase 9; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cytochromes c; Down-Regulation; Drug Therapy, Combination; Glioblastoma; Humans; Paclitaxel; Proto-Oncogene Proteins c-bcl-2; Valproic Acid

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψ(m) and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψ(m) decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.

Topics: Acetylcysteine; Adenosine Triphosphate; Apoptosis; Cell Line, Tumor; Cytochromes c; Enzyme Inhibitors; Free Radical Scavengers; Glioblastoma; Glycolysis; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction; Proteolysis; Pyruvates; Reactive Oxygen Species

The effect of the biologically active prenylated chalcone and potential anticancer agent xanthohumol (1) has been investigated on apoptosis of the T98G human malignant glioblastoma cell line. Compound 1 decreased the viability of T98G cells by induction of apoptosis in a time- and concentration-dependent manner. Apoptosis induced by 1 was associated with activation of caspase-3, caspase-9, and PARP cleavage and was mediated by the mitochondrial pathway, as exemplified by mitochondrial depolarization, cytochrome c release, and downregulation of the antiapoptotic Bcl-2 protein. Xanthohumol induced intracellular reactive oxygen species (ROS), an effect that was reduced by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC). Intracellular ROS production appeared essential for the activation of the mitochondrial pathway and induction of apoptosis after exposure to 1. Oxidative stress due to treatment with 1 was associated with MAPK activation, as determined by ERK1/2 and p38 phosphorylation. Phosphorylation of ERK1/2 and p38 was attenuated using NAC to inhibit ROS production. After treatment with 1, ROS provided a specific environment that resulted in MAPK-induced cell death, with this effect reduced by the ERK1/2 specific inhibitor PD98059 and partially inhibited by the p38 inhibitor SB203580. These findings suggest that xanthohumol (1) is a potential chemotherapeutic agent for the treatment of glioblastoma multiforme.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cyclohexenes; Cytochromes c; Dose-Response Relationship, Drug; Flavonoids; Glioblastoma; Humans; Imidazoles; Italy; Mitogen-Activated Protein Kinases; Molecular Structure; Poly(ADP-ribose) Polymerases; Propiophenones; Pyridines; Reactive Oxygen Species; Terpenes

A major concern of cancer chemotherapy is the side effects caused by the non-specific targeting of both normal and cancerous cells by therapeutic drugs. Much emphasis has been placed on discovering new compounds that target tumour cells more efficiently and selectively with minimal toxic effects on normal cells.. The cytotoxic effect of thymoquinone, a component derived from the plant Nigella sativa, was tested on human glioblastoma and normal cells. Our findings demonstrated that glioblastoma cells were more sensitive to thymoquinone-induced antiproliferative effects. Thymoquinone induced DNA damage, cell cycle arrest and apoptosis in the glioblastoma cells. It was also observed that thymoquinone facilitated telomere attrition by inhibiting the activity of telomerase. In addition to these, we investigated the role of DNA-PKcs on thymoquinone mediated changes in telomere length. Telomeres in glioblastoma cells with DNA-PKcs were more sensitive to thymoquinone mediated effects as compared to those cells deficient in DNA-PKcs.. Our results indicate that thymoquinone induces DNA damage, telomere attrition by inhibiting telomerase and cell death in glioblastoma cells. Telomere shortening was found to be dependent on the status of DNA-PKcs. Collectively, these data suggest that thymoquinone could be useful as a potential chemotherapeutic agent in the management for brain tumours.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Benzoquinones; Brain; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Damage; DNA Repair; DNA-Activated Protein Kinase; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Nuclear Proteins; Telomerase; Telomere

We previously elucidated an important role for gangliosides in renal cell carcinoma-mediated T lymphocyte apoptosis, although the mechanism by which they mediated lymphocyte death remained unclear. Here, we show that when added in purified form, GD3 is internalized by activated T cells, initiating a series of proapoptotic events, including the induction of reactive oxygen species (ROS), an enhancement of p53 and Bax accumulation, an increase in mitochondrial permeability, cytochrome c release, and the activation of caspase-9. GD3-induced apoptosis of activated T cells was dose dependent and inhibitable by pretreating the lymphocytes with N-acetylcysteine, cyclosporin A, or bongkrekic acid, emphasizing the essential role of ROS and mitochondrial permeability to the process. Ganglioside-induced T-cell killing was associated with the caspase-dependent degradation of nuclear factor-kappaB-inducible, antiapoptotic proteins, including RelA; this suggests that their loss is initiated only after the cascade is activated and that their disappearance amplifies but not triggers GD3 susceptibility. Resting T cells did not internalize appreciable levels of GD3 and did not undergo any of the proapoptotic changes that characterize activated T lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the role of the intact transcription factor in mediating protection from the ganglioside.

Topics: Acetylcysteine; Antioxidants; Apoptosis; Carcinoma, Renal Cell; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; Gangliosides; Glioblastoma; Humans; Jurkat Cells; Kidney Neoplasms; Lymphocyte Activation; Mitochondrial Membranes; Reactive Oxygen Species; T-Lymphocytes

The anti-neoplastic drug taxol binds to beta-tubulin to prevent tumor cell division, promoting cell death. However, high dose taxol treatment may induce cell death in normal cells too. The anti-apoptotic molecule Bcl-2 is upregulated in many cancer cells to protect them from apoptosis. In the current study, we knocked down Bcl-2 expression using cognate siRNA during low-dose taxol treatment to induce apoptosis in two human glioblastoma U138MG and U251MG cell lines. The cells were treated with either 100 nM taxol or 100 nM Bcl-2 siRNA or both for 72 h. Immunofluorescent stainings for calpain and active caspase-3 showed increases in expression and co-localization of these proteases in apoptotic cells. Fluorometric assays demonstrated increases in intracellular free [Ca(2+)], calpain, and caspase-3 indicating augmentation of apoptosis. Western blotting demonstrated dramatic increases in the levels of Bax, Bak, tBid, active caspases, DNA fragmentation factor-40 (DFF40), cleaved fragments of lamin, fodrin, and poly(ADP-ribose) polymerase (PARP) during apoptosis. The events related to apoptosis were prominent more in combination therapy than in either treatment alone. Our current study demonstrated that Bcl-2 siRNA significantly augmented taxol mediated apoptosis in different human glioblastoma cells through induction of calpain and caspase proteolytic activities. Thus, combination of taxol and Bcl-2 siRNA offers a novel therapeutic strategy for controlling the malignant growth of human glioblastoma cells.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calcium; Calpain; Caspase 3; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Fluorescent Antibody Technique; Glioblastoma; Humans; In Situ Nick-End Labeling; Paclitaxel; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a synthetic retinoid that has shown biological activity against several malignant tumors and minimal side effects in humans. To explore the mechanisms underlying the chemotherapeutic effects of 4-HPR in glioblastoma, we used two human glioblastoma T98G and U87MG cell lines. In situ methylene blue staining showed the morphological features of astrocytic differentiation in glioblastoma cells following exposure to 1 microM and 2 microM 4-HPR for a short duration (24 h). Astrocytic differentiation was associated with an increase in expression of glial fibrillary acidic protein (GFAP) and downregulation of telomerase. Wright staining and ApopTag assay indicated appearance of apoptotic features in glioblastoma cells following exposure to 1 microM and 2 microM 4-HPR for a long duration (72 h). We found that 4-HPR caused apoptosis with activation of caspase-8 and cleavage of Bid to truncated Bid (tBid). Besides, apoptosis was associated with alterations in expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and Smac, downregulation of selective baculoviral inhibitor-of-apoptosis repeat containing (BIRC) molecules, an increase in intracellular free [Ca2+], and activation of calpain and caspase-3. Taken together, these results strongly suggested that 4-HPR could be used at low doses for induction of both differentiation and apoptosis in human glioblastoma cells.

Topics: Analysis of Variance; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Astrocytes; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Brain Neoplasms; Calpain; Caspase 3; Caspase 8; Cell Differentiation; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Fenretinide; Glial Fibrillary Acidic Protein; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Telomerase

We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway.. With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm.. The human glioblastoma cell lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment.. This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3.

Topics: Antineoplastic Agents; Apoptosis; Benzodiazepinones; Blotting, Western; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Erucic Acids; Glioblastoma; Humans; Isoquinolines; Ligands; Membrane Potential, Mitochondrial; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Phosphorylcholine; Receptors, GABA

Glioblastoma is the most malignant brain tumor in humans and an average survival of glioblastoma patients hardly exceeds 12 months. Taxol is a plant-derived anti-cancer agent, which has been used in the treatments of many solid tumors. Deletion or mutation of phosphatase and tension homolog located on chromosome ten (PTEN) occurs in more than 80% of glioblastomas. We examined the sensitivity of human glioblastoma LN18 (PTEN-positive) and A172 (PTEN-negative) cells to Taxol for induction of apoptosis. Wright staining showed morphological features of apoptosis after treatment with different doses of Taxol for 24 h. Significant amount of apoptosis occurred in LN18 cells after treatment with 25 nM Taxol, while in A172 cells only after treatment with 50 nM Taxol. Western blotting with an antibody that could specifically detect activation or phosphorylation of Akt (p-Akt) did not show any p-Akt in LN18 cells but an increase in p-Akt in A172 cells. Activation of Akt in A172 cells could be reversed by pre-treatment of the cells with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002, indicating involvement of PI3K activity in this process. Apoptosis occurred with an increase in Bax:Bcl-2 and mitochondrial release of cytochrome c into the cytosol leading to activation of mitochondria-dependent caspase cascade. Taxol did not cause upregulation of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, in LN18 cells but substantial upregulation of VEGF in A172 cells. After treatment with Taxol, increases in p-Akt and VEGF could maintain survival and angiogenesis, respectively, in PTEN-negative glioblastoma. As a single chemotherapy, Taxol might be more efficacious in PTEN-positive glioblastoma than in PTEN-negative glioblastoma. Thus, our study showed differential sensitivity of PTEN-positive and PTEN-negative glioblastoma cells to Taxol.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Glioblastoma; Humans; Mitochondria; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Signal Transduction; Vascular Endothelial Growth Factor A

Deletion or mutation of phosphatase and tensin homolog located on chromosome ten (PTEN) occurs in as high as 80% glioblastoma. All-trans retinoic acid (ATRA) induces differentiation in cancer cells. Interferon-gamma (IFN-gamma) induces apoptosis in many cancers including glioblastoma. We used the combination of ATRA and IFN-gamma to control growth of human glioblastoma LN18 (PTEN-proficient) and U87MG (PTEN-deficient) cells and explored any advantage of having PTEN in the cells.. LN18 and U87MG cells were treated with ATRA (1 microM) for 7 days and then IFN-gamma (5 ng/ml) for 1 day. Methylene blue staining indicated astrocytic differentiation. Wright staining and ApopTag assay showed characteristic features of apoptosis. Western blotting demonstrated the levels of specific proteins.. ATRA and IFN-gamma alone and in combination could induce apoptosis in LN18 cells; while ATRA alone induced differentiation only, IFN-gamma alone induced apoptosis, and ATRA plus IFN-gamma increased apoptosis in U87MG cells. The variation in induction of apoptosis by ATRA alone might be attributed to difference in PTEN expression in the two cell lines. Compared with control cells, IFN-gamma alone and ATRA plus IFN-gamma increased PTEN expression in LN18 cells while there was no PTEN expression or induction in U87MG cells after treatments with ATRA alone and ATRA plus IFN-gamma. Apoptosis in both cell lines was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c into the cytosol, and calpain and caspase-3 activities. Treatments elevated p27(kip1) and decreased CDK2 levels in both cell lines, indicating cell cycle arrest at G(1)/S phase.. The combination of ATRA and IFN-gamma could control the growth of both PTEN-proficient and PTEN-deficient glioblastoma cells by arresting cell division and inducing differentiation and apoptosis. Thus, our study indicated that the growth of both PTEN-proficient and PTEN-deficient glioblastoma cells could effectively be controlled by treatment with the combination of ATRA and IFN-gamma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Astrocytes; Blotting, Western; Cell Culture Techniques; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Cytosol; DNA Fragmentation; Down-Regulation; Glioblastoma; Humans; In Situ Nick-End Labeling; Interferon-gamma; Mitochondria; PTEN Phosphohydrolase; Recombinant Proteins; Tretinoin; Up-Regulation

Gambogic acid (GA) is the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia. The present study aims to demonstrate that gambogic acid (GA) has potent anticancer activity for glioblastoma by in vitro and in vivo study. Rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood-brain barrier (BBB). To reveal an involvement of the intrinsic mitochondrial pathway of apoptosis, the mitochondrial membrane potential and the western blot evaluation of Bax, Bcl-2, Caspase-3, caspase-9 and cytochrome c released from mitochondria were performed. Angiogenesis was detected by CD31 immunochemical study. The results showed that the uptake of GA by rBMEC was time-dependent, which indicated that it could pass BBB and represent a possible new target in glioma therapy. GA could cause apoptosis of rat C6 glioma cells in vitro in a concentration-dependent manner by triggering the intrinsic mitochondrial pathway of apoptosis. In vivo study also revealed that i.v. injection of GA once a day for two weeks could significantly reduce tumor volumes by antiangiogenesis and apoptotic induction of glioma cells. Collectively, the current data indicated that GA may be of potential use in treatment of glioblastoma by apoptotic induction and antiangiogenic effects.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Cytochromes c; Glioblastoma; Membrane Potential, Mitochondrial; Rats; Rats, Sprague-Dawley; Tumor Burden; Xanthones

Unlike oleate and linoleate, palmitate induced mitochondrial apoptosis in GL15 glioblastoma cells. Decrease in membrane potential in a subpopulation of mitochondria of palmitate-treated cells was revealed using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide probe. The diminished ability to reduce a tetrazolium salt indicated an impairment of mitochondrial function. Up to 50% cytochrome c (cyt c) was detached from the inner mitochondrial membrane and released outside mitochondria in palmitate-treated cells, whereas no release was detected after oleate and linoleate treatments. Cyt c release into the cytosol was followed by caspase 3 activation. Released cyt c and caspase 3 activity were not affected by neutral and acid sphingomyelinase inhibitors and by the inhibitor of serine palmitoyltransferase cycloserine, indicating that apoptosis was independent of the ceramide pathway, nor the mitochondrial pro-apoptotic AIF or Bcl-2/Bax factors appeared to be involved in the effect. Utilization of palmitate by GL15 cells altered phospholipid composition. Cardiolipin (CL), the lipid involved in cyt c interaction with the inner mitochondrial membrane, was decreased and highly saturated. This produced an imbalance in hydrophilic/hydrophobic interactions underlying the anchorage of cyt c, by weakening the hydrophobic component and facilitating detachment of the protein and activation of downstream processes. The primary role of CL was explored by supplying GL15 with exogenous CL through a fusion process of CL liposomes with cell plasma membrane. Fused CL moved to mitochondria, as detected by nonylacridine orange probe. Enrichment of mitochondrial membranes with CL prior to palmitate treatment of cells caused decreased cyt c release and caspase 3 activity.

Topics: Apoptosis; Brain Neoplasms; Cardiolipins; Caspase 3; Cell Line, Tumor; Cytochromes c; Energy Metabolism; Enzyme Activation; Glioblastoma; Humans; Indicators and Reagents; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction; Oxidative Stress; Palmitates; Protein Transport; Tetrazolium Salts

The HIPPI (HIP-1 protein interactor) protein is a multifunctional protein that is involved in the regulation of apoptosis. The interaction partners of HIPPI include HIP-1 (Huntingtin-interacting protein-1), Apoptin, Homer1c, Rybp/DEDAF, and BAR (bifunctional apoptosis regulator). In search for other binding partners of HIPPI, we performed a yeast two hybrid screen and identified BLOC1S2 (Biogenesis of lysosome-related organelles complex-1 subunit 2) as a novel HIPPI-interacting protein. In co-immunoprecipitation assays, BLOC1S2 specifically associates with HIPPI, but not with HIP-1. To study the expression of BLOC1S2 on the protein level, we generated a mouse monoclonal antibody specific for BLOC1S2 and a multiple tissue array comprising 70 normal and cancer tissue samples of diverse origin. BLOC1S2 protein is widely expressed in normal tissue as well as in malignant tumors with a tendency towards lower expression levels in certain subtypes of tumors. On the subcellular level, BLOC1S2 is expressed in an organellar-like pattern and co-localizes with mitochondria. Over-expression of BLOC1S2 in the presence or absence of HIPPI does not induce apoptosis. However, BLOC1S2 and HIPPI sensitize NCH89 glioblastoma cells to the pro-apoptotic actions of staurosporine and the death ligand TRAIL by enhancing caspase activation, cytochrome c release, and disruption of the mitochondrial membrane potential. Given its interaction with HIPPI and its pro-apoptotic activity, BLOC1S2 might play an important functional role in cancer and neurodegenerative diseases.

Topics: Adaptor Proteins, Signal Transducing; Adult; Amino Acid Sequence; Animals; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Cytochromes c; Female; Glioblastoma; HeLa Cells; Humans; Immunohistochemistry; Male; Membrane Potential, Mitochondrial; Mitochondria; Molecular Sequence Data; Neoplasms; Protein Binding; Proteins; Sequence Alignment; Staurosporine; Tissue Distribution; TNF-Related Apoptosis-Inducing Ligand

Glioblastoma (GBM) is an astrocytic brain tumor characterized by an aggressive clinical course and intense resistance to all therapeutic modalities. Here, we report the identification and functional characterization of Bcl2L12 (Bcl2-like-12) that is robustly expressed in nearly all human primary GBMs examined. Enforced Bcl2L12 expression confers marked apoptosis resistance in primary cortical astrocytes, and, conversely, its RNA interference (RNAi)-mediated knockdown sensitizes human glioma cell lines toward apoptosis in vitro and impairs tumor growth with increased intratumoral apoptosis in vivo. Mechanistically, Bcl2L12 expression does not affect cytochrome c release or apoptosome-driven caspase-9 activation, but instead inhibits post-mitochondrial apoptosis signaling at the level of effector caspase activation. One of Bcl2L12's mechanisms of action stems from its ability to interact with and neutralize caspase-7. Notably, while enforced Bcl2L12 expression inhibits apoptosis, it also engenders a pronecrotic state, which mirrors the cellular phenotype elicited by genetic or pharmacologic inhibition of post-mitochondrial apoptosis molecules. Thus, Bcl2L12 contributes to the classical tumor biological features of GBM such as intense apoptosis resistance and florid necrosis, and may provide a target for enhanced therapeutic responsiveness of this lethal cancer.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Apoptosomes; Astrocytes; Brain Neoplasms; Caspase 7; Caspase 9; Cytochromes c; Enzyme Activation; Glioblastoma; Glioma; Humans; Immunoglobulin G; Mice; Mice, SCID; Mitochondria; Muscle Proteins; Necrosis; Proteins; Proto-Oncogene Proteins c-bcl-2; Rabbits; RNA, Small Interfering; Signal Transduction

Glioblastoma is the most malignant human brain tumor that shows poor response to existing therapeutic agents. Search continues for an effective therapy for controlling this deadliest brain tumor. Curcumin (CCM), a polyphenolic compound from Curcuma longa, possesses anti-cancer properties in both in vitro and in vivo. In the present investigation, we evaluated the therapeutic efficacy of CCM against human malignant glioblastoma U87MG cells. Trypan blue dye exclusion test showed decreased viability of U87MG cells with increasing dose of CCM. Wright staining and ApopTag assay, respectively, showed the morphological and biochemical features of apoptosis in U87MG cells treated with 25 microM and 50 microM of CCM for 24 h. Western blotting showed activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and release of cytochrome c from mitochondria followed by activation of caspase-9 and caspase-3 for apoptosis. Also, CCM treatments increased cytosolic level of Smac/Diablo to suppress the inhibitor-of-apoptosis proteins and down regulated anti-apoptotic nuclear factor kappa B (NFkappaB), favoring the apoptosis. Increased activities of calpain and caspase-3 cleaved 270 kDa alpha-spectrin at specific sites generating 145 kDa spectrin break down product (SBDP) and 120 kDa SBDP, respectively, leading to apoptosis in U87MG cells. Results show that CCM is an effective therapeutic agent for suppression of anti-apoptotic factors and activation of calpain and caspase proteolytic cascades for apoptosis in human malignant glioblastoma cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Brain Neoplasms; Caspase 8; Cell Line, Tumor; Cell Survival; Curcumin; Cysteine Endopeptidases; Cytochromes c; Cytosol; DNA Fragmentation; Enzyme Activation; Glioblastoma; Humans; Mitochondria; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Spectrin; Trypan Blue

Glioblastoma multiforme (GBM) is one of the most therapeutically refractory human cancers. Elevated cellular polyamine levels are a common feature of cancer cells, including GBM cells, and the polyamine pathway has been explored as a potential therapeutic target to inhibit polyamine biosynthesis or activate polyamine catabolism. In this study, we investigated the effect of N1,N11-diethyl-norspermine (DENSPM), a spermine analog that activates polyamine catabolism, in GBM cells. The in vitro cell culture experiments showed that DENSPM increased the sub-G1 apoptotic cell population in GBM cell lines but caused minimal cytotoxicity in normal astrocytes. Prior to apoptosis induction, DENSPM caused the elevation of spermidine/spermine N1-acetyltransferase (SSAT) expression accompanied by a decrease in polyamine levels and an increase of acetylated polyamine levels, which temporally coincided with the onset of hydrogen peroxide (H2O2) induction in the cells. The cytotoxic effects of DENSPM in the GBM cells could be partially attenuated by either turning down SSAT mRNA with small interference RNA or inhibiting H2O2 production with N1-acetylpolymine oxidase (APAO)/spermine oxidase (SMO) inhibitor. Though mitochondrial damage was induced, neither activation of the caspase cascade nor cytochrome c redistribution between the mitochondria and cytoplasm was observed. Systemic DENSPM treatment of mice with intracerebral GBM led to longer survival. Taken together, our studies indicate that DENSPM kills GBM cells through induction of SSAT coupled with H2O2 production, which is a potential target for GBM therapy.

Topics: Animals; Cell Death; Cytochromes c; Glioblastoma; Humans; Hydrogen Peroxide; Male; Membrane Potentials; Mice; Mice, Nude; Mitochondria; Polyamines; RNA, Small Interfering; Spermine

Phosphatase and tension homolog located on chromosome ten (PTEN) is a tumor suppressor as it negatively regulates activation of Akt. Mutation or deletion of PTEN has been found in as high as 80% of glioblastomas, which harbor aberrant cell signaling passing through the phosphatidylinositol-3-kinase (PI3K) and Akt (PI3K/Akt) survival pathway. Glioblastoma cells without functional PTEN are not easily amenable to apoptosis. We investigated the possibility of modulation of signal transduction pathways for induction of apoptosis in human glioblastoma T98G (PTEN-harboring) and U87MG (PTEN-deficient) cell lines after treatment with the combination of all-trans retinoic acid (ATRA) and interferon-gamma (IFN-gamma). Treatment with ATRA plus IFN-gamma stimulated PTEN expression and suppressed Akt activation in T98G cells, whereas no PTEN expression but Akt activation in U87MG cells under the same conditions. Pretreatment of U87MG cells with the PI3K inhibitor LY294002 could prevent Akt activation. Interestingly, ATRA plus IFN-gamma could significantly decrease cell viability and increase morphological features of apoptosis in both cell lines. Combination of ATRA and IFN-gamma showed more efficacy than IFN-gamma alone in causing apoptosis that occurred due to increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and caspase-3 activity. Luciferase reporter gene assay showed that combination of ATRA and IFN-gamma significantly down regulated transcriptional activity of the nuclear factor kappa B (NF-kappaB), a survival signaling factor, in U87MG cells. Thus, combination of ATRA and IFN-gamma caused significant amounts of apoptosis in T98G cells due to suppression of the PI3K/Akt survival pathway while the same treatment caused apoptosis in U87MG cells due to down regulation of the NF-kappaB activity. Therefore, the combination of ATRA and IFN-gamma could modulate different survival signal transduction pathways for induction of apoptosis and should be considered as an effective therapeutic strategy for controlling the growth of both PTEN-harboring and PTEN-deficient glioblastomas.

Topics: Apoptosis; bcl-2-Associated X Protein; Brain Neoplasms; Cell Survival; Cytochromes c; Cytosol; Genes, Reporter; Glioblastoma; Humans; Interferon-gamma; Luciferases; Nerve Tissue Proteins; NF-kappa B; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Recombinant Proteins; Signal Transduction; Transfection; Tretinoin

Glioblastoma is the deadliest brain tumor in humans. Current therapies are mostly ineffective and new agents need to be explored for controlling this devastating disease. Inositol hexaphosphate (IP6) is a phytochemical that is widely found in corns, cereals, nuts, and high fiber-content foods. Previous studies demonstrated anti-cancer properties of IP6 in several in vitro and in vivo tumor models. However, therapeutic efficacy of IP6 has not yet been evaluated in glioblastoma. Here, we explored the molecular mechanism of action of IP6 in human malignant glioblastoma T98G cells. The viability of T98G cells decreased following treatment with increasing doses of IP6. T98G cells exposed to 0.25, 0.5, and 1 mM IP6 for 24 h showed morphological and biochemical features of apoptosis. Western blotting indicated changes in expression of Bax and Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio and upregulation of cytosolic levels of cytochrome c and Smac/Diablo, suggesting involvement of mitochondria-dependent caspase cascade in apoptosis. IP6 downregulated cell survival factors such as baculovirus inhibitor-of-apoptosis repeat containing-2 (BIRC-2) protein and telomerase to promote apoptosis. Upregulation of calpain and caspase-9 occurred in course of apoptosis. Increased activities of calpain and caspase-3 cleaved 270 kD alpha-spectrin at specific sites generating 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Increased caspase-3 activity also cleaved inhibitor of caspase-3-activated DNase and poly(ADP-ribose) polymerase. Collectively, our results demonstrated that IP6 down regulated the survival factors BIRC-2 and telomerase and upregulated calpain and caspase-3 activities for apoptosis in T98G cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Brain Neoplasms; Calpain; Caspases; Cell Line, Tumor; Cytochromes c; Cytosol; DNA Fragmentation; Enzyme Induction; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; Nerve Tissue Proteins; Phytic Acid; Proto-Oncogene Proteins c-bcl-2; Trypan Blue; Ubiquitin-Protein Ligases

A prominent feature of glioblastoma is its resistance to death receptor-mediated apoptosis. In this study, we explored the possibility of modulating death receptor-induced cell death with the c-Jun-NH2-terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and inducing "ribotoxic stress." We found that anisomycin and death receptor ligand anti-Fas antibody CH-11 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induce apoptosis in multiple human glioblastoma cell lines. For example, in U87 cells, anisomycin reduced the IC50 of CH-11 by more than 20-fold (from 500 to 25 ng/mL). Cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P<0.001), respectively. Anisomycin and TRAIL were found to be similarly synergistic in glioblastoma cells maintained as tumor xenografts. The potentiation of death receptor-dependent cell death by anisomycin was specific because emetine, another ribosome inhibitor that does not induce ribotoxic stress or activate JNK, did not have a similar effect. Synergistic cell death was predominantly apoptotic involving both extrinsic and intrinsic pathways. Expression of Fas, FasL, FLIP, and Fas-associated death domain (FADD) was not changed following treatment with anisomycin+CH-11. JNK was activated 10- to 22-fold by anisomycin+CH-11 in U87 cells. Inhibiting JNK activation with pharmacologic inhibitors of JNKK and JNK or with dominant negative mitogen-activated protein kinase (MAPK) kinase kinase 2 (MEKK2) significantly prevented cell death induced by the combination of anisomycin+CH-11. We further found that anisomycin+CH-11 up-regulated the proapoptotic protein Bim by approximately 14-fold. Simultaneously inhibiting Bim expression and JNK activation additively desensitized U87 cells to anisomycin+CH-11. These findings show that anisomycin-induced ribotoxic stress sensitizes glioblastoma cells to death receptor-induced apoptosis via a specific mechanism requiring both JNK activation and Bim induction.

Topics: Anisomycin; Antibodies; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Blotting, Western; Caspases; Cell Cycle; Cell Proliferation; Cytochromes c; Drug Synergism; Fas Ligand Protein; fas Receptor; Flow Cytometry; Glioblastoma; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Oxidative Stress; Proto-Oncogene Proteins; Receptors, Death Domain; Ribosomes; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

Cisplatin is a DNA-damaging chemotherapeutic drug that may have a role in the adjuvant chemotherapy of several solid tumors, such as malignant glioblastoma, and the status of p53 tumor suppressor protein is a critical determinant of cisplatin chemosensitivity. In the present study, we showed the relationship of p53 status and chemosensitivity of cisplatin between two human malignant glioblastoma cell lines, A172 and T98G, harboring wild-type and mutant-type p53, respectively. Cisplatin was found to be more cytotoxic to A172 than T98G cells in a time- and concentration-dependent manner. Cisplatin-induced cytotoxicity manifested as apoptosis, characterized by genomic DNA fragmentation, nuclear condensation and an increase in sub-G1 population. Cisplatin induced the accumulation of p53 and p21 proteins in A172 cells, but not in T98G cells. The introduction of the adenovirus-mediated wild-type p53 gene into T98G cells resulted in the decrease of viability as well as the increase in sub-G1 population with p53 accumulation, activation of caspase-3 protease and release of cytochrome c from the mitochondria. These data strongly suggest that the expression of p53 is essential for the cytotoxic effect of cisplatin in human malignant glioblastoma cells, A172 and T98G, and the introduction of apoptotic signal molecules, such as p53, will be beneficial to achieve chemosensitivity in malignant glioma.

Topics: Apoptosis; Brain Neoplasms; Caspase 3; Caspase 9; Caspases; Cisplatin; Cytochromes c; DNA Damage; Enzyme Activation; Genes, p53; Glioblastoma; Humans; Transduction, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

A new approach for an amperometric array sensor platform employing arrays of sensors in a 24-well cell culture plate format has been developed for simultaneous in vitro determination of nitric oxide (NO) and superoxide free radicals (O(2)(-)) produced by stimulated cells. The work reported focuses on the direct, real-time monitoring of extracellular production of these two analytes, as well as the effects of their interaction. The sensor platform was manufactured by a combination of sputtering gold electrodes and screen-printing carbon electrodes. The O(2)(-) sensor uses covalent immobilization of cytochrome c via a binder, DTSSP (3,3'-dithio-bis(sulphosuccinimidylpropionate) onto the surface of the Au electrodes, whereas the NO sensor system involves an NiTSPc (nickel tetrasulfonated phthalocyanine) film electrodeposited onto the surface of the carbon electrodes and subsequently covered with an external layer of Nafion. For in vitro demonstration of the platforms as a potential drug-screening system, A172 glioblastoma cells were cultured and transferred into the 24-well arrays. Simultaneous and direct monitoring of NO and O(2)(-) production as a response to chemicals of biomedical relevance was carried out. The results obtained demonstrated that it would be possible to envisage a drug screening platform for compounds designed to be inhibitors of nitric oxide synthase or to have an inhibitory effect on superoxide free radical production. By suitable modification of the electrodes employed it would also be possible to extend the platform to measure alternative species.

Topics: Biosensing Techniques; Cell Culture Techniques; Cell Line, Tumor; Computer Systems; Cytochromes c; Electrochemistry; Equipment Design; Equipment Failure Analysis; Glioblastoma; Humans; Nitric Oxide; Superoxides; Systems Integration; Transducers

The X-chromosome-linked inhibitor of apoptosis protein (XIAP) prevents apoptosis from activated transmembrane death receptors and confers tumour resistance to irradiation and chemotherapy. Despite the important oncologic implications, data concerning glioblastoma in this regard are few and isolated. The objective of this study was to examine the role of XIAP in the signalling pathway of TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis in chemoresistant human glioblastoma cells.. Downregulators of XIAP, low-dose cisplatin, etoposide (VP 16) or second mitochondria-derived activator of caspase (Smac)-Tat peptide, were applied to 2 chemoresistant glioblastoma cell lines of fresh isolates to identify the impact of these sensitizing agents on the cytotoxicity of TRAIL. Hoechst staining for apoptotic nuclear morphology and Western blot analysis for the corresponding levels of proteins that regulate apoptotic pathways including XIAP were performed. The involvement of mitochondrial pathways marked by the release of cytochrome c or Smac/direct IAP (inhibitor of apoptosis protein)-binding protein with low P1 (DIABLO), or both, was assessed by confocal fluorescence microscopy.. Downregulators of XIAP induced apoptosis in a dose-dependent manner with TRAIL in 1 chemoresistant glioblastoma cell line. Here, XIAP downregulation modulated by Smac-Tat peptide resulted in increased TRAIL-induced cell death. In addition, TRAIL was shown to enhance the translocation of Smac/DIABLO from mitochondria to the cytosol in cells that underwent apoptosis, which in turn neutralized XIAP activity. In comparison, the second chemoresistant glioblastoma cell line showed no regulatory XIAP effect. This finding correlates with the upstream effect of mutant p53 and BCL-X(L) status that were upregulated in this chemoresistant cell line.. These results support the use of selective or tailored therapeutic strategies that synergistically sensitize chemoresistant glioblastoma to TRAIL-mediated apoptosis by administering appropriate XIAP downregulating agents.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Carrier Proteins; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Cisplatin; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Etoposide; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Mitochondria; Mitochondrial Proteins; Proteins; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha; X-Linked Inhibitor of Apoptosis Protein