cytochrome-c-t and Brain-Neoplasms

Cell death associated with mitochondrial dysfunction is common in acute neurological disorders and in neurodegenerative diseases. Neuronal apoptosis is regulated by multiple proteins, including neuroglobin, a small heme protein of ancient origin. Neuroglobin is found in high concentration in some neurons, and its high expression has been shown to promote survival of neurons in vitro and to protect brain from damage by both stroke and Alzheimer's disease in vivo. Early studies suggested this protective role might arise from the protein's capacity to bind oxygen or react with nitric oxide. Recent data, however, suggests that neither of these functions is likely to be of physiological significance. Other studies have shown that neuroglobin reacts very rapidly with cytochrome c released from mitochondria during cell death, thus interfering with the intrinsic pathway of apoptosis. Systems level computational modelling suggests that the physiological role of neuroglobin is to reset the trigger level for the post-mitochondrial execution of apoptosis. An understanding of the mechanism of action of neuroglobin might thus provide a rational basis for the design of new drug targets for inhibiting excessive neuronal cell death.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Death; Cytochromes c; Globins; Humans; Mitochondria; Nerve Tissue Proteins; Neuroglobin; Neuroprotective Agents; Protein Binding; Signal Transduction

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

The present study aimed to investigate the effects of cold atmospheric plasma (CAP)‑activated Ringer's solution on osteosarcoma cell lines MG63 and U2OS, and to identify the molecular mechanism underlying these effects. CAP‑activated Ringer's solution was used to treat osteosarcoma cell lines MG63 and U2OS for 30 min. Cell viability was measured using the MTT method. The apoptosis rate was detected using Annexin‑V and propidium iodide. The expression levels of cytochrome c, caspase‑3 and polyADP ribose polymerase (PARP) in MG63 cells were analyzed via western blotting. The change in mitochondrial membrane potential was detected via the JC‑1 dye method and verified by the level of reactive oxygen species (ROS). CAP‑activated Ringer's solution inhibited the proliferation of MG63 and U2OS cells in a dose‑ and time‑dependent manner. Furthermore, CAP‑activated Ringer's solution induced the apoptosis of MG63 cells, increased the intracellular ROS level, decreased the mitochondrial membrane potential level, and induced the release of cytochrome c. CAP‑activated Ringer's solution inhibits osteosarcoma cell proliferation through intracellular ROS‑mediated mitochondrial apoptosis.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Plasma Gases; Reactive Oxygen Species; Ringer's Solution

Malignant gliomas are the most lethal form of primary brain tumors. Despite advances in cancer therapy, the prognosis of glioma patients has remained poor. Cytochrome c (Cytc), an endogenous heme-based protein, holds tremendous potential to treat gliomas because of its innate capacity to trigger apoptosis. To this end, a hybrid cytochrome c-chlorotoxin (Cytc-CTX) protein was biosynthesized to enable cellular uptake of the cell impenetrable Cytc using CTX transporters. A nucleotide sequence containing 1 : 1 Cytc and CTX was constructed and separated by a hexahistidine-tag and an enterokinase cleavage site. The sequence was cloned into a pBTR1 plasmid, expressed in Escherichia coli, purified via 2-dimensional chromatography. The identity and size of the protein were determined by Western blot and mass spectrometry. Cytc in this soluble hybrid protein has similar structure and stability as human Cytc and the hybrid protein is endocytosed into a glioma cell line, while displaying potent cytotoxicity and a favorable therapeutic index. Its facile, low-cost, and high yield synthesis, biocompatibility, and robustness suggest that the hybrid protein is a promising candidate for antiglioma drug evaluation.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Survival; Cells, Cultured; Cytochromes c; Dose-Response Relationship, Drug; Glioma; Humans; Scorpion Venoms; Structure-Activity Relationship

In this study, we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to some gliomas. Using the techniques of confocal imaging, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and small interfering (siRNA) knockdown against the PCFT, we demonstrated that Gl261 and A172 glioma cells, but not U87 and primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by cell death. The FA-Cyt c NPs (100 µg/mL), had no cytotoxic effects in astrocytes but caused death in glioma cells, according to their level of expression of PCFT. Whole-cell patch clamp recording revealed FA-induced membrane currents in FA-Cyt c NPs-sensitive gliomas, that were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a route for internalization of FA-conjugated NPs in these glioma cells. Analysis of human glioblastoma specimens revealed that at least 25% of glioblastomas express elevated level of either PCFT or folate receptor (FOLR1). We conclude that the PCFT provides a mechanism for targeted delivery of drugs to some gliomas as a starting point for the development of efficient methods for treating gliomas with high expression of PCFT and/or FOLR1.

Topics: Animals; Astrocytes; Brain Neoplasms; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Folic Acid; Glioma; Humans; Mice; Mice, Inbred C57BL; Nanoconjugates; Proton-Coupled Folate Transporter

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

Dihydrotanshinone, a functional food in China, is an effective anti-cardiovascular disease substance isolated from Salvia miltiorrhiza (S. miltiorrhiza). Glioma is considered to be fatal due to its poor prognosis and few effective therapeutic options. In this study, we investigated the anticancer effects of S. miltiorrhiza extract dihydrotanshinone on human glioma SHG-44 cells, by using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay, Hoechst 33258 nuclear staining, Annexin V/propidium iodide double staining, as well as western blot analysis. The results showed that dihydrotanshinone effectively suppressed SHG-44 cells proliferation and induced apoptosis in both dose- and time-dependent manner. Moreover, we demonstrated that dihydrotanshinone increased the activation of caspases (caspase-3 and caspase-9) and the release of cytochrome c in SHG-44 cells. Overall, dihydrotanshinone could induce apoptosis and inhibit proliferation of glioma cells by regulating caspases and cytochrome c. This study suggests that dihydrotanshinone may serve as a potential treatment option for patients with glioma.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Glioma; Humans; Plant Extracts; Salvia miltiorrhiza

Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor-associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma-associated brain endothelial cells. In the present study, we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma-associated brain endothelial cells. Annexin-V/PI staining showed that T11TS treatment in glioma-induced rats increases apoptosis of glioma-associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC-1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma-associated brain endothelial cells. Flowcytometry, immunoblotting, and in situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment-upmodulated expression of p53, Bax, Fas, FasL, and FADD in glioma associated endothelial cells and downregulated Bcl-2 protein. T11TS therapy induced cytochrome-c release into cytosol, activated caspase -9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma-associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas-dependent extrinsic pathway. The pro-apoptotic action of T11TS on glioma-associated endothelial cells provides crucial insight into how T11TS exerts its anti-angiogenic function in glioma. J. Cell. Physiol. 232: 526-539, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Brain Neoplasms; Caspases; Cytochromes c; Cytosol; Endothelial Cells; Enzyme Activation; Fas Ligand Protein; fas Receptor; Fas-Associated Death Domain Protein; Female; Glioma; Glycopeptides; Male; Membrane Glycoproteins; Membrane Potential, Mitochondrial; Models, Biological; Neovascularization, Pathologic; Rats; Sheep; Tumor Suppressor Protein p53

Gambogic acid (GA), a natural product with a xanthone structure, has a broad range of anti-proliferative effects on cancer cell lines. We evaluated GA for its cytotoxic effects on T98G glioblastoma cells. GA exhibited potent anti-proliferative activity and induced apoptosis in T98G glioblastoma cells in a dose-dependent manner. Incubation of cells with GA revealed apoptotic features including increased Bax and AIF expression, cytochrome c release, and cleavage of caspase-3, -8, -9, and PARP, while Bcl-2 expression was downregulated. Furthermore, GA induced reactive oxygen species (ROS) generation in T98G cells. Our results indicate that GA increases Bax- and AIF-associated apoptotic signaling in glioblastoma cells.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Glioma; Humans; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Xanthones

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

To study the relationship of basic fibroblast growth factor (bFGF) and signal transducer and activator of transcription 3(STAT3) in glioma apoptosis and possible mechanisms of its interaction.. Two glioblastomamultiforme (GBM) cell lines: U87 (wild-type p53) and U251 (mutant p53) were used in this study and divided into normal control group, mock group and experiment group.Small interfering RNA-carried recombinant lentivirus, LV-bFGFsiRNA and LV-STAT3siRNA, targeting bFGF and STAT3 were constructed respectively. After 48 hours of lentivirus transfection, small molecular inhibitors were used to block specific signaling pathways, AG490 20 µmol/L blocking JAK, LY294002 20 µmol/L blocking PI3K/Akt pathways for 24 hours, 48 hours and 72 hours, respectively. Then, apoptosis, changes in apoptosis-related proteins and mitochondrial membrane potential were detected through the methods of flow cytometry, protein chip and confocal microscopy, respectively.Groups were compared using single factor analysis of variance (One-way ANOVA).. Western blot results revealed the levels of Tyr705 and Ser727 phosphorylationin reduced in a time dependent manner by blocking JAK and PI3K/Akt pathway respectively. The results of flow cytometry showed that the apoptosis rate in normal control group, mock group, experiment group were 17.97% ± 0.24%, 18.26% ± 0.88%, 46.57% ± 1.63% in U87 cells and 15.94% ± 1.18%, 16.88% ± 0.17%, 39.34% ± 0.87% in U251 cells, respectively. There was no statistically significant change between the normal control group and the mock group (P > 0.05) , while when compared with the experiment group, both group showed statistically significant difference (F = 697.41, 729.58, both P < 0.05). The results of protein chip demonstrated that protein expression of Bad, Caspase3, Cytochrome C, p27 were higher and XIAP was lower in the experiment group compared with the normal control group and mock group. Also, confocal microscopy could detect apoptosis and mitochondrial membrane potential reduced significantly in the experimental group compared with the normal control group and the mock group.. bFGF mainly interacts with STAT3 tyrosine site-pSTAT3(Tyr705) to influence the level of STAT3 phosphorylation;blocking bFGF/STAT3 signaling pathway can induce glioma cell apoptosis through mitochondrial pathway.

Topics: Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cytochromes c; Fibroblast Growth Factor 2; Glioma; Humans; Mitochondria; Phosphatidylinositol 3-Kinases; Phosphorylation; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transfection; Tyrphostins

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

Glioblastoma multiforme (GBM) is the most aggressive astrocytoma, and therapeutic options are generally limited to surgical resection, radiotherapy, and Temozolomide (TMZ) chemotherapy. TMZ is a DNA alkylating agent that causes DNA damage and induces cell death. Unfortunately, glioma cells often develop resistance to TMZ treatment, with DNA de-methylation of the MGMT promoter identified as the primary reason. However, the contributions from proteins that normally protect cells against cytotoxic stress in TMZ-induced apoptosis have not been extensively explored. Here, we showed that increasing the level of the gap junction protein, Cx43, in human LN18 and LN229 glioma cells enhances resistance to TMZ treatment while knockdown of Cx43 in these same cells sensitizes them to TMZ treatment. By expressing a channel-dead or a C-terminal truncation mutant of Cx43, we show that Cx43-mediated TMZ resistance involves both channel dependent and independent functions. Expression of Cx43 in LN229 cells decreases TMZ-induced apoptosis, as determined by Annexin V staining. Cx43-mediated chemoresistance appears to be acting via a mitochondrial apoptosis pathway as manifested by the reduction in Bax/Bcl-2 ratio and the release of cytochrome C. Our findings highlight additional mechanisms and proteins that contribute to TMZ resistance, and raise the possibility of increasing TMZ efficiency by targeting Cx43 protein. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Topics: Analysis of Variance; Annexin A5; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Connexin 43; Cytochromes c; Dacarbazine; Dose-Response Relationship, Drug; Fluoresceins; Glioma; Humans; Mitochondria; Mutation; Phosphorylation; RNA, Small Interfering; Signal Transduction; Temozolomide; Time Factors; Transfection

Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.

Topics: Antineoplastic Agents; Brain Neoplasms; Caspase 3; Cell Death; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Glioma; Humans; Mitochondria; Necrosis; Photochemotherapy; Photosensitizing Agents; Porphyrins

Growth-inhibitory effects of mimosine, a plant amino acid, on rat C6 glioma cells were analyzed. Mimosine markedly inhibited proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent manner. Mimosine-mediated apoptosis was accompanied by promoting reactive oxygen species (ROS) generation in mitochondria, and by decreased mitochondrial membrane potential (Δψ), and release of cytochrome c from mitochondria, followed by caspase 3 activation. Furthermore, mimosine increased the phosphorylation level of c-Jun-N-terminal protein kinase and p38, which was the downstream effect of ROS accumulation. Mimosine was confirmed to show profound effects on apoptosis of C6 glioma cells by ROS-regulated mitochondria pathway, and these results bear on the hypothesized potential for mimosine as promising agents in the treatment of malignant gliomas.

Topics: Apoptosis; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 7; Cell Line, Tumor; Comet Assay; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Fluorescent Antibody Technique; Glioma; Humans; MAP Kinase Kinase 4; Membrane Potentials; Mimosine; Mitochondria; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species

A previous study showed that miR-221/222 can regulate cell apoptosis. p53 is a well known tumor suppressor which can influence the chemosensitivity of glioma cells. However, the effect of miR-221/222 in gliomas with different p53 status is unknown. Here, we demostrate that knockdown of miR-221/222 increases apoptosis in human gliomas of different p53 types (U251 cells, p53 mutant-type; LN308 cells, p53 null-type; and U87 cells, p53 wild-type). Furthermore, the effect of miR-221/22 caused no change of p53 expression in the glioma cells studied. In addition, when a specific siRNA against p53 was employed in U87 cells, no attenuation of apoptosis was found after knockdown of miR-221/222. Importantly, we found that As-miR-221/222-treated cells increased expression of Bax, cytochrome c, Apaf-1 and cleaved-caspase-3. Our results showed that low expression of miR-221/222 sensitized glioma cells to temozolomide (TMZ); in addition, ectopic expression of PUMA by pcDNA-PUMA had a similar effect. Taken together, our study indicates that downregulated miR-221/222 can sensitize glioma cells to TMZ by regulating apoptosis independently of p53 status.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Brain Neoplasms; Caspase 3; Cytochromes c; Dacarbazine; Down-Regulation; Gene Knockdown Techniques; Glioma; Humans; MicroRNAs; Mitochondria; Proto-Oncogene Proteins; RNA, Small Interfering; Signal Transduction; Temozolomide; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Blood-Brain Barrier; Brain Neoplasms; Caspases; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cytochromes c; DNA Fragmentation; Endothelial Cells; Humans; Lignans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred ICR; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Tight Junctions

Guanidino compounds, as methylguanidine (MG), may play an important role in the etiology of neurological complications which occur in uremic syndrome. Dementia is a neurological complication more common in uremic patients than in general population and several types of dementia are associated to astroglial apoptosis. Here we report the effect of MG on oxidative stress-induced apoptosis in rat glioma cell line (C6) in vitro. The oxidative stress was induced by hydrogen peroxide (H(2)O(2); 1 mM) and the cellular and molecular parameters were observed after 18 h. Uremic conditions were simulated by pre-incubation of C6 cells with MG (0.1-10 mM) 1h before H(2)O(2)-induced oxidative stress. MG alone did not affect cell viability, but it significantly increased cell death induced by H(2)O(2), as assessed by MTT assay. This effect could be related to the MG capability to enhance H(2)O(2) pro-apoptotic effect on C6 cells. The fluorescent dye FURA 2-AM test showed a significant raise in [Ca(2+)](i) in MG and H(2)O(2) co-treated C6 cells, mainly for depolarizing mitochondrial membrane potential. Furthermore, MG in a concentration-dependent manner, significantly increased H(2)O(2)-induced Bax expression, activation of caspase-3 and PARP in C6 cells. This study firstly reports that the uremic catabolyte MG could contribute to neurodegeneration associated to uremia enhancing the pro-apoptotic effect of H(2)O(2) and through an alteration in mitochondrial calcium homeostasis in glial cells.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Blotting, Western; Brain Neoplasms; Calcium Signaling; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Glioma; Hydrogen Peroxide; Indicators and Reagents; Methylguanidine; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Propidium; Rats

beta-Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that beta-elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of beta-elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of beta-elemene on prostate cancer cell death was mediated through the induction of apoptosis.. The MTT assay was used to evaluate the ability of beta-elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA-based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl-2, caspases, cytochrome c and poly(ADP-ribose) polymerase (PARP) were analysed by Western blotting.. Here, we showed that beta-elemene had an antiproliferative effect on androgen-insensitive prostate carcinoma DU145 and PC-3 cells. Treatment with beta-elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of beta-elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 microg/ml (230-465 microm). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by beta-elemene in a dose- and time-dependent manner. Moreover, beta-elemene exposure resulted in a decreased Bcl-2 protein level, increased cytochrome c release, and activated PARP and caspase-3, -7, -9, and -10 in prostate cancer cells.. Overall, these findings suggest that beta-elemene exerts broad-spectrum antitumour activity against many types of solid carcinoma and supports a proposal of beta-elemene as a new potentially therapeutic drug for castration-resistant prostate cancer and other solid tumours.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Lung Neoplasms; Male; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Time Factors; Uterine Cervical Neoplasms

Glioblastoma multiforme is the most frequent and aggressive primary brain tumor. A strong rationale to identify innovative approaches to treat these tumors is required since treatment failures result in local recurrences and median survivals range from 9 to 12 months. Glioma cells are reported to have less mitochondrial content compared to adjacent normal brain cells. Based on this difference, we suggest a new strategy, utilizing protection of normal brain cells by mitochondria-targeted electron scavengers and antioxidants-nitroxides-thus allowing for the escalation of the radiation doses. In this paper, we report that a conjugate of nitroxide with a hydrophobic cation, triphenyl-phosphonium (TPEY-Tempo), significantly protected brain endothelial cells from γ-irradiation-induced apoptosis while radiosensitizing brain tumor cells. Thus, TPEY-Tempo may be a promising adjunct in the treatment of glioblastoma with the potential to not only prolong survival but also to maintain quality of life and reduce treatment toxicity.

Topics: Apoptosis; Brain; Brain Neoplasms; Caspase 3; Cell Line, Transformed; Cyclic N-Oxides; Cytochromes c; Dose-Response Relationship, Radiation; Electron Transport Complex IV; Endothelial Cells; Gamma Rays; Glioma; Humans; Mitochondria; Neuroprotective Agents; Nitrogen Oxides; Organic Chemicals; Organoselenium Compounds

Glioblastoma represent the most common primary brain tumor in adults and are currently considered incurable. We investigated antiproliferative and anti-invasive mechanisms of 6-OH-11-O-hydroxyfenantrene (IIF), a retinoid X receptor ligand, and pioglitazone (PGZ), a peroxisome proliferator-activated receptor gamma activator, in three different glioblastoma cell lines. A dose-dependent reduction of tumor invasion and strong decrease of matrix metalloproteinases 2 and 9 expression was observed, especially when a combination therapy of IIF and PGZ was administered. Combined treatment also markedly reduced proliferation and induced apoptosis in all glioma cell lines tested. This was in particular accompanied by decrease of antiapoptotic proteins Bcl2 and p53, while simultaneously pro-apoptotic cytochrome c, cleaved caspase 3, Bax and Bad levels increased. These in vitro findings were further substantiated in a murine glioma model in vivo, where oral administration of PGZ and IIF resulted in significantly reduced tumor volume and proliferation. Of note, treatment with nuclear receptor ligands was not only effective when the treatment was initiated shortly after the intraparenchymal seeding of the glioma cells, but even when initiated in the last third of the observation period. Collectively, our results demonstrate the effectiveness of a combined treatment of ligands of proliferator-activated receptor and retinoid X receptor against glioblastoma.

Topics: Analysis of Variance; Animals; Annexin A5; Antineoplastic Agents; bcl-2-Associated X Protein; Brain Neoplasms; Bromodeoxyuridine; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Glioma; Humans; Ligands; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Pioglitazone; PPAR gamma; Proto-Oncogene Proteins c-bcl-2; Rats; Retinoid X Receptor gamma; Tetrazolium Salts; Thiazoles; Thiazolidinediones; Transfection; Tretinoin; Tumor Stem Cell Assay

Gliomas, particularly glioblastomas, are among the deadliest of human tumors. Gliomas emerge through the accumulation of recurrent chromosomal alterations, some of which target yet-to-be-discovered cancer genes. A persistent question concerns the biological basis for the coselection of these alterations during gliomagenesis.. To describe a network model of a cooperative genetic landscape in gliomas and to evaluate its clinical relevance.. Multidimensional genomic profiles and clinical profiles of 501 patients with gliomas (45 tumors in an initial discovery set collected between 2001 and 2004 and 456 tumors in validation sets made public between 2006 and 2008) from multiple academic centers in the United States and The Cancer Genome Atlas Pilot Project (TCGA).. Identification of genes with coincident genetic alterations, correlated gene dosage and gene expression, and multiple functional interactions; association between those genes and patient survival.. Gliomas select for a nonrandom genetic landscape-a consistent pattern of chromosomal alterations-that involves altered regions ("territories") on chromosomes 1p, 7, 8q, 9p, 10, 12q, 13q, 19q, 20, and 22q (false-discovery rate-corrected P<.05). A network model shows that these territories harbor genes with putative synergistic, tumor-promoting relationships. The coalteration of the most interactive of these genes in glioblastoma is associated with unfavorable patient survival. A multigene risk scoring model based on 7 landscape genes (POLD2, CYCS, MYC, AKR1C3, YME1L1, ANXA7, and PDCD4) is associated with the duration of overall survival in 189 glioblastoma samples from TCGA (global log-rank P = .02 comparing 3 survival curves for patients with 0-2, 3-4, and 5-7 dosage-altered genes). Groups of patients with 0 to 2 (low-risk group) and 5 to 7 (high-risk group) dosage-altered genes experienced 49.24 and 79.56 deaths per 100 person-years (hazard ratio [HR], 1.63; 95% confidence interval [CI], 1.10-2.40; Cox regression model P = .02), respectively. These associations with survival are validated using gene expression data in 3 independent glioma studies, comprising 76 (global log-rank P = .003; 47.89 vs 15.13 deaths per 100 person-years for high risk vs low risk; Cox model HR, 3.04; 95% CI, 1.49-6.20; P = .002) and 70 (global log-rank P = .008; 83.43 vs 16.14 deaths per 100 person-years for high risk vs low risk; HR, 3.86; 95% CI, 1.59-9.35; P = .003) high-grade gliomas and 191 glioblastomas (global log-rank P = .002; 83.23 vs 34.16 deaths per 100 person-years for high risk vs low risk; HR, 2.27; 95% CI, 1.44-3.58; P<.001).. The alteration of multiple networking genes by recurrent chromosomal aberrations in gliomas deregulates critical signaling pathways through multiple, cooperative mechanisms. These mutations, which are likely due to nonrandom selection of a distinct genetic landscape during gliomagenesis, are associated with patient prognosis.

Topics: 3-Hydroxysteroid Dehydrogenases; Aldo-Keto Reductase Family 1 Member C3; Annexin A7; Apoptosis Regulatory Proteins; ATPases Associated with Diverse Cellular Activities; Brain Neoplasms; Chromosome Aberrations; Cytochromes c; Dosage Compensation, Genetic; Epistasis, Genetic; Female; Gene Dosage; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Neoplasm; Genome-Wide Association Study; Glioma; Humans; Hydroxyprostaglandin Dehydrogenases; Male; Metalloendopeptidases; Middle Aged; Mitochondrial Proteins; Models, Genetic; Mutation; Nuclear Proteins; Prognosis; Proportional Hazards Models; Risk; RNA-Binding Proteins; Signal Transduction; Survival Analysis

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

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.

Topics: Adult; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Brain Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Mitochondria; Protein Kinase C; Proto-Oncogene Proteins c-akt; Receptors, TNF-Related Apoptosis-Inducing Ligand

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

To evaluate the association of signals of apoptosis namely, TGF-beta1, TNF-alpha and cytochrome c release in cytoplasm with survival rate to determine the potential use of such parameters as predictive markers for patients with astrocytomas.. We measured TGF-beta1, TNF-alpha and cytoplasmic cytochrome c in 30 astrocytic tumors Grade II, III and IV.. We found that TNF-alpha and cytochrome c release in Grade IV tends to be significantly lower than those in Grade II, whereas TGF-beta1 did not significantly change in the different grades. Patients with astrocytic tumors having elevated cytochrome c showed a better survival rate compared to those with less release. There is neither a correlation shown between TNF-alpha and cytochrome c release nor between TNF-alpha and patient survival. TGF-beta1 was positively correlated with cytochrome c release. Patients showing such correlation had increased survival rate over 18 months follow up period.. These data suggest that TGF-beta1 and cytochrome c may be useful prognostic markers that help patients' stratification and in adjusting the disciplines of therapy.

Topics: Adolescent; Adult; Aged; Apoptosis; Astrocytoma; Biomarkers, Tumor; Brain Neoplasms; Child; Child, Preschool; Cytochromes c; Female; Humans; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

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

The basic mechanism of cell death induced by 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) (ALA-PDT) in glioma cells has not been fully elucidated. In this study, the details of the cell death mechanism induced by ALA-PDT were investigated in three human glioma cell lines (U251MG, U87MG, and U118MG) in vitro. To evaluate the manner of accumulation of protoporphyrin IX (PpIX), intracellular PpIX contents were measured by flow cytometry after incubation with 5-ALA. To analyze the mechanism of cell death, U251MG cells were assayed by the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling (TUNEL) method, and the caspase activity was measured after ALA-PDT. Furthermore, the mitochondrial membrane potential (MMP) and the release of mitochondrial cytochrome c were determined. PpIX fluorescence reached a plateau 4 h after exposure to 5-ALA. The proportion of dead cells increased with increases in the dosage of light. These cells were confirmed by TUNEL staining to be apoptotic. Increases in the activity of both caspase-3 and -9, a decrease in MMP, and a marked increase in cytochrome c in the cytosolic fraction were found after cells were subjected to PDT. These results indicate that a dysfunction of MMP is followed by mitochondrial cytochrome c release, which triggers apoptosis through a mitochondrial pathway. ALA-PDT induces massive apoptosis due to the direct activation of a mitochondrial pathway, which is resistant to many anti-apoptotic processes, in human glioma cells. This finding implies that ALA-PDT is a promising therapy for the treatment of apoptosis-reluctant tumors such as malignant gliomas.

Topics: Aminolevulinic Acid; Apoptosis; Brain Neoplasms; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Glioma; Humans; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Photochemotherapy; Photosensitizing Agents; Protoporphyrins; 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

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

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Astrocytoma; Brain; Brain Neoplasms; Caspases; Cytochromes c; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Medulloblastoma; Neurons; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Transcription, Genetic

Cannabinoids have been associated with tumor regression and apoptosis of cancer cells. Recently, we have shown that R(+)-methanandamide (R(+)-MA) induces apoptosis of H4 human neuroglioma cells via a mechanism involving de novo expression of the cyclooxygenase-2 (COX-2) enzyme. The present study investigated a possible involvement of a mitochondrial-driven pathway in this process.. Cell death was determined by the WST-1 cell viability test, and changes in apoptotic parameters [i.e., release of mitochondrial cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase (PARP)] were detected by Western blotting.. H4 cells treated with R(+)-MA showed typical signs of mitochondrial apoptosis, i.e., release of mitochondrial cytochrome c into the cytosol and activation of initiator caspase-9. Moreover, activation of the executor caspase-3 was observed following cannabinoid treatment. Cells were fully protected from apoptotic cell death by the caspase-3 inhibitor Ac-DEVD-CHO, indicating a crucial role for caspase-3 activation in R(+)-MA-elicited apoptosis. Furthermore, cleavage of the caspase-3 target protein PARP was registered. All of the aforementioned effects were substantially reduced by the selective COX-2 inhibitor celecoxib (1 muM) at a pharmacologically relevant, nonapoptotic concentration.. R(+)-MA-induced apoptosis is mediated via a mitochondrial-dependent pathway that becomes activated, at least in part, through up-regulation of the COX-2 enzyme.

Topics: Animals; Apoptosis; Arachidonic Acids; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 9; Caspases; Celecoxib; Cell Survival; CHO Cells; Cricetinae; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytochromes c; Glioma; Humans; Mitochondria; Poly(ADP-ribose) Polymerases; Pyrazoles; Signal Transduction; Sulfonamides

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 preferential loss of brain cholinergic neurons in the course of Alzheimer's disease and other encephalopathies is accompanied by a proportional impairment of acetyl-CoA synthesizing capacity in affected brains. Particular susceptibility of cholinergic neurons to neurodegeneration might results from insufficient supply of acetyl-CoA for energy production and acetylcholine synthesis in these conditions. Exposure of SN56 cholinergic neuroblastoma cells to dibutyryl cAMP and retinoic acid for 3 days caused their morphologic differentiation along with the increase in choline acetyltransferase activity, acetylcholine content and release, calcium content, and the expression of p75 neurotrophin receptors. Acetyl-CoA content correlated inversely with choline acetyltransferase activity in different lines of SN56 cells. In differentiated cells, aluminum (1 mM), amyloid beta(25-35) (0.001 mM), and sodium nitroprusside (1 mM), caused much greater decrease of pyruvate dehydrogenase and choline acetyltransferase activities and cell viability than in nondifferentiated ones. Aluminum (1 mM) aggravated suppressory effects of amyloid beta on choline acetyltransferase and pyruvate dehydrogenase activities and viability of differentiated cells. Similar additive inhibitory effects were observed upon combined exposure of differentiated cells to sodium nitroprusside and amyloid beta(25-35). None or much smaller suppressory effects of these neurotoxins were observed in nondifferentiated cells. Increase in the fraction of nonviable differentiated cells positively correlated with losses of choline acetyltransferase, pyruvate dehydrogenase activities, and cytoplasmic cytochrome c content in different neurotoxic conditions. These data indicate that highly differentiated cholinergic neurons may be more susceptible to aluminum and other neurotoxins than the nondifferentiated ones due to relative shortage of acetyl-CoA, increased content of Ca(2+), and expression of p75 receptors, yielding increase in cytoplasmic cytochrome c and subsequently grater rate of death of the former ones.

Topics: Acetyl Coenzyme A; Acetylcholine; Aluminum; Amyloid beta-Peptides; Animals; Autonomic Nervous System Diseases; Brain Neoplasms; Calcium; Cell Differentiation; Cell Line, Tumor; Choline O-Acetyltransferase; Cytochromes c; DNA, Complementary; Energy Metabolism; Immunohistochemistry; Mice; Neuroblastoma; Neurotoxins; Nitroprusside; Peptide Fragments; Phenotype; Rats; Receptor, trkA; Trypan Blue

Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.

Topics: Antifungal Agents; Apoptosis; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 9; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Humans; L-Lactate Dehydrogenase; Microscopy, Fluorescence; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Subcellular Fractions; Tetrazolium Salts; Thiazoles; Thimerosal

Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear.. We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests.. Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006).. Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cardiolipins; Caspases; Coenzyme A Ligases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Female; Flavoproteins; Gene Transfer Techniques; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasms, Experimental; Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Stomach Neoplasms; Transfection; Transplantation, Heterologous; Triazenes; Tumor Suppressor Protein p53

Malignant glioma cells are generally resistant or only weakly sensitive to tumor necrosis factor family of cell death-inducing ligands, including TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L. The chemopreventive activity of polyphenolic compounds present in plant-derived food products has been well recognized in epidemiological studies; however, the mechanism of chemoprevention by these dietary constituents largely remains unknown. Curcumin, the yellow pigment in the spice turmeric, has profound anti-inflammatory activity and exhibits chemopreventive and tumor growth inhibitory activity. In the present study, we investigated whether curcumin sensitizes malignant glioma cell lines U251MG and U87MG to TRAIL-induced apoptosis. Treatment with low concentrations (5-20 microM) of curcumin alone had no effect on the viability of either cell line. At low concentration (5 ng/ml) TRAIL induced cytotoxicity in U251MG cells but not in U87MG cells. Whereas curcumin at subtoxic concentration sensitized U87MG cells to TRAIL-induced cytotoxicity, it had no effect on TRAIL-mediated cytotoxicity in U251MG cells. The combined curcumin and TRAIL treatment enhanced accumulation of hypo-diploid U87MG cells in sub G1 cell cycle phase and induced the cleavage of procaspases-3, -8, -9 and release of cytochrome c from mitochondria. These data indicate that curcumin differentially sensitizes glioma cells to TRAIL-induced apoptosis through the activation of both extrinsic (receptor-mediated) and intrinsic (chemical-induced) pathways of apoptosis. These results define a potential use of curcumin to sensitize glioma cells for TRAIL-mediated immunotherapy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Curcumin; Cytochromes c; Enzyme Activation; Glioma; Humans; Indicators and Reagents; L-Lactate Dehydrogenase; Membrane Glycoproteins; Mitochondria; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Fifty percent of high-grade glioma patients die within a year of diagnosis and less than two percent survive five years postdiagnosis. Elucidating apoptosis signaling pathways may assist in designing better adjuvant therapies. Preliminary characterizations suggested that glioma cells may either employ mitochondrial-independent or -dependent death receptor-induced apoptotic pathways, characteristic of cells termed type I and type II, respectively. In the present study, we generated panels of clonal transfectants overexpressing various levels of Bcl-2, in two parental glioma cell lines. These cells were used to explore molecular factors determining the necessity for mitochondrial amplification of death receptor signaling. Moderate Bcl-2 expression was sufficient to render one glioma cell line (D270) resistant to apoptosis induced by Fas ligand or TRAIL, consistent with these cells being type II. However, expression of even very high levels of Bcl-2 in a second line (D645) did not affect death ligand sensitivity, indicative of a type I phenotype. D270 cells expressed much less caspase-8 protein than D645 cells. Enforced overexpression of caspase-8 (or cytoplasmic Diablo/Smac) in D270 cells overcame Bcl-2 inhibition of death ligand-induced apoptosis, converting them from type II to type I. This indicates that caspase-8 levels can influence the requirement for mitochondrial involvement in death receptor apoptotic signaling in glioma cells.

Topics: Apoptosis; Base Sequence; Brain Neoplasms; Caspase 8; Caspases; Cell Line, Tumor; Cytochromes c; DNA Primers; Glioma; Humans; Mitochondria; Substrate Specificity

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

Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death.

Topics: Apoptosis; Brain Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; DNA, Mitochondrial; DNA, Neoplasm; Dose-Response Relationship, Drug; Electron Transport; Enzyme Activation; Fluorometry; Humans; Hydrogen Peroxide; In Situ Nick-End Labeling; Microscopy, Confocal; Mitochondria; Neoplasm Proteins; Oxidants; Teratocarcinoma

In the hyperthermal treatment, the wild type (wt) p53 plays an important role in apoptosis induction in the tumor cells. In human gliomas, p53 frequently has some form of mutation. The mutant type (mt) p53 does not work properly as a tumor suppressor and this may result in poor responses during treatment. We investigated the relationship between apoptosis-inducing factor (AIF) and apoptosis under various thermal conditions (43, 45, and 47 degrees C for 1 h) using four p53-wild or -mutant human glioma cell lines (A172, T98G, U251MG, and YKG-1). AIF translocation from the mitochondria to the nucleus under hyperthermal conditions was demonstrated by confocal laser microscopy. The percentage of AIF-positive nuclei increased significantly in comparison with the control in all cell lines and in all temperature groups except for YKG-1 at 47 degrees C. Immunoblot analyses of the nuclear fraction of each cell line revealed temperature-dependent increases in AIF. A simultaneous release of cytochrome c from the mitochondria to the cytosol was noted. A flow cytometric analysis showed that apoptosis induction occurred more often in a temperature-dependent manner in the 45 and 47 degrees C groups than in the control group. These findings indicate that the hyperthermal conditions can lead to AIF translocation and apoptotic cell death in the p53-mutant human glioma cells. The present report is the first description of AIF-induced apoptosis in hyperthermia.

Topics: Apoptosis; Apoptosis Inducing Factor; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Flavoproteins; Flow Cytometry; Genes, p53; Glioma; Humans; Hyperthermia, Induced; Immunoblotting; Immunohistochemistry; Membrane Proteins; Microscopy, Confocal; Mutation; Protein Transport

Hypoxia induces apoptosis in primary and transformed cells and in various tumor cell lines in vitro. In contrast, there is little apoptosis and predominant necrosis despite extensive hypoxia in human glioblastomas in vivo. We here characterize ultrastructural and biochemical features of cell death in LN-229, LN-18 and U87MG malignant glioma cells in a paradigm of hypoxia with partial glucose deprivation in vitro. Electron microscopic analysis of hypoxia-challenged glioma cells demonstrated early stages of apoptosis but predominant necrosis. ATP levels declined during hypoxia, but recovered with re-exposure to normoxic conditions unless hypoxia exceeded 8 h. Longer hypoxic exposure resulted in irreversible ATP depletion and delayed cell death. Hypoxia induced mitochondrial release of cytochrome c, but there was no cleavage of caspases 3, 7, 8 or 9, and no DNA fragmentation. Ectopic expression of BCL-XL conferred protection from hypoxia-induced cell death, whereas the overexpression of the antiapoptotic proteins X-linked-inhibitor-of-apoptosis-protein and cytokine response modifier-A had no effect. These findings suggest that glioma cells resist adverse effects of hypoxia until energy stores are depleted and then undergo necrosis rather than apoptosis because of energy deprivation.

Topics: Adenosine Triphosphate; bcl-X Protein; Brain Neoplasms; Caspases; Cell Death; Cell Hypoxia; Cell Nucleus; Cytochromes c; Cytoplasm; Energy Metabolism; Glioma; Glucose; Humans; Immunologic Factors; Microscopy, Electron; Mitochondria; Necrosis; Proteins; Proto-Oncogene Proteins c-bcl-2; Starvation; Tumor Cells, Cultured; X-Linked Inhibitor of Apoptosis Protein

Many malignant glioma cells express death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), yet some of these cells are resistant to TRAIL. Here, we examined signaling events in TRAIL-induced apoptosis and searched for therapeutic agents that could overcome TRAIL resistance in glioma cells. TRAIL induced apoptosis through death receptor 5 (DR5) and was mediated by caspase-8-initiated extrinsic and intrinsic mitochondrial pathways in sensitive glioma cell lines. TRAIL also triggered apoptosis in resistant glioma cell lines through the same pathways, but only if the cells were pretreated with chemotherapeutic agents, cisplatin, camptothecin and etoposide. Previous studies suggested that this was due to an increase in DR5 expression in wild-type TP53 cells, but this mechanism did not account for cells with mutant TP53. Here, we show that a more general effect of these agents is to downregulate caspase-8 inhibitor c-FLIP(S) (the short form of cellular Fas-associated death domain-fike interleukin-1-converting enzyme-inhibitory protein) and up-regulate Bak, a pro-apoptotic Bcl-2 family member, independently of cell's TP53 status. Furthermore, we showed that TRAIL alone or in combination with chemotherapeutic agents, induced apoptosis in primary tumor cultures from patients with malignant gliomas, reinforcing the potential of TRAIL as an effective therapeutic agent for malignant gliomas.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; Blotting, Northern; Blotting, Western; Brain Neoplasms; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Flavoproteins; Flow Cytometry; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; Proteins; Receptors, Tumor Necrosis Factor; rho GTP-Binding Proteins; Signal Transduction; Subcellular Fractions; Temperature; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein

We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria.

Topics: Androstadienes; Brain Neoplasms; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Fractionation; Cell Line; Cell Membrane; Cell Nucleus; Cytochromes c; Cytosol; Drug Interactions; Electron Transport Complex IV; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoblotting; Insulin-Like Growth Factor I; Ionophores; Kidney; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Porins; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Serine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Superoxide Dismutase; Threonine; Time Factors; Voltage-Dependent Anion Channel 1; Wortmannin