calpain and Glioblastoma

Recent studies have reported the important roles of dopamine receptors in the early development and progression of glioblastoma (GBM). The present research aimed to explore the antineoplastic effect and intrinsic pathways of action of dopamine receptor D1 agonist SKF83959 on GBM cells. Flow cytometric analysis revealed a significant level of apoptotic cell death under SKF83959 treatment. SKF83959 administration increased intracellular calcium levels and oxidative stress through the phospholipase C/inositol trisphosphate pathway. The downstream calpains were activated and dysregulated by the increased calcium levels. The mitochondrial membrane potential‑dependent staining assay revealed decreased mitochondrial transmembrane potential in GBM cells under SKF83959 treatment. The mitochondrial/cytosolic fraction and western blotting further demonstrated mitochondrial dysfunction and endoplasmic reticulum stress, followed by apoptosis. The calpain inhibitor, calpastatin, significantly reversed the increase in mitochondrial injury and endoplasmic reticulum stress and eventually ameliorated GBM cell apoptosis during SKF83959 treatment. Finally, the

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Aged; Animals; Apoptosis; Brain; Brain Neoplasms; Calpain; Cell Line, Tumor; Chemotherapy, Adjuvant; Endoplasmic Reticulum Stress; Female; Glioblastoma; Humans; Male; Membrane Potential, Mitochondrial; Middle Aged; Mitochondria; Neurosurgical Procedures; Receptors, Dopamine D1; Signal Transduction; Xenograft Model Antitumor Assays

Filamin A is the most widely expressed isoform of filamin in mammalian tissues. It can be hydrolyzed by Calpain, producing a 90-kDa carboxyl-terminal fragment (ABP90). Calpeptin is a chemical inhibitor of Calpain, which can inhibit this effect. It has been shown that ABP90 acts as a transcription factor which is involved in mediating cell signaling. However, the significance of ABP90 and its clinical signature with underlying mechanisms have not been well studied in glioblastoma multiforme (GBM).. ABP90 protein was measured in 36 glioma patients by Western blot. Human GBM cell lines U87 and A172 were used to clarify the precise role of ABP90. CCK-8 assay was used to analyze the cell viability. Transwell invasion assay and wound healing assay were used to analyze the migration and invasion. Expression of matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot.. ABP90 protein expression was lower in GBM tissues. The patients with low ABP90 protein expression had a shorter OS time (p = 0.046). After being treated with Calpain, the expression of ABP90 was upregulated, which led to a decline of cell viability, enhanced the efficacy of temozolomide and restrained the cell invasion. Calpeptin could inhibit the effect. The mechanism might be involved in the balance of MMP2/TIMP2.. Our present data suggest that ABP90 expression is a significant prognostic factor and may play an important role in cell viability, chemotherapeutic sensitivity and invasion of GBM.

Topics: Biomarkers, Tumor; Brain Neoplasms; Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dipeptides; Filamins; Glioblastoma; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Prognosis; Temozolomide; Tissue Inhibitor of Metalloproteinase-2

Glioblastoma (GBM) is a brain tumor that remains largely incurable because of its highly-infiltrative properties. Nuclear factor I (NFI)-type transcription factors regulate genes associated with GBM cell migration and infiltration. We have previously shown that NFI activity depends on the NFI phosphorylation state and that calcineurin phosphatase dephosphorylates and activates NFI. Calcineurin is cleaved and activated by calpain proteases whose activity is, in turn, regulated by an endogenous inhibitor, calpastatin (CAST). The

Topics: Calpain; Cell Line, Tumor; Cell Movement; Glioblastoma; Humans; NFI Transcription Factors

Glioblastoma multiforme is one of the most malignant tumors of the human central nervous system characterized by high degree of invasiveness. Focusing on this invasive nature, we investigated whether ganglioside-specific sialidase NEU3 might be involved, because gangliosides are major components of brain tissues, and cell surface sialic acids, as target residues of sialidase catalysis, are thought to be closely related to cell invasion.. NEU3 mRNA levels of human glioblastoma specimens were evaluated by quantitative RT-PCR. Human glioblastoma cell lines, U251, A172, and T98G were used for cell invasion and migration by transwell and cell scratching assay. The molecules involved in the signaling cascade were investigated by western blot and immunofluorescent microscopy.. NEU3 expression was down-regulated in human glioblastoma specimens. In the human glioblastoma cell lines, NEU3 overexpression reduced invasion and migration by promoting the assembly of focal adhesions through reduced calpain-dependent proteolysis, but NEU3 silencing resulted in accelerating cell invasion via disassembly of focal adhesions. In NEU3-silenced cells, elevation of calpain activity and GM3 accumulation were observed, as results of reduced sialidase hydrolysis, localization of calpain and GM3 at the cell lamellipodium being demonstrated by immunofluorescence microscopy.. Sialidase NEU3 was found to exert a great influence on cell invasion in regulation of calpain activity and focal adhesion disassembly and consequent invasive potential of glioblastoma cells.. This first demonstration of sialidase involvement in invasive potential of gliolastoma cells may point to NEU3 as an attractive treatment target of human gliomas.

Topics: Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Focal Adhesions; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Neoplasm Invasiveness; Neuraminidase; Proteolysis

miR-124 and Capn4 are aberrantly expressed in glioblastoma multiforme (GBM) tissues. In the present study, we investigated miR-124 and Capn4 expression in GBM tissue specimens. The role of miR-124 and Capn4 in the migration and invasion of glioma cells in vitro was also examined. miR-124 and Capn4 expression in 20 GBM and 6 control brain specimens was examined using RT-qPCR and immuno-blotting. Data from The Cancer Genome Atlas were retrieved. Candidate mRNA target sites of miR-124 were predicted using TargetScan/microRNA and binding was examined using dual luciferase reporter assays. The U87 and U251 cells were transfected with scrambled microRNA, miR-124 mimics and/or pLenti-Capn4 prior to wound‑healing and Transwell invasion assays. Proteins involved in the epithelial-mesenchymal transition were examined using immunoblotting. The results showed that miR-124 was significantly downregulated in GBM tissues. Immunoblotting showed a marked upregulation of Capn4 expression in GBM tissues. The Spearman's correlation analysis revealed a negative association between miR-124 expression and Capn4 protein levels. TargetScan/microRNA predicted the miR-124 binding site in the nucleotide 440-446 region within the Capn4 3'-UTR, which was confirmed by luciferase assays. Wound‑healing and Transwell invasion assays demonstrated that Capn4 downregulation or miR-124 mimics suppressed the migration and invasion of glioma cells. Capn4 downregulation or miR-124 mimics reduced the level of phospho-FAK and MMP2, vimentin and N-cadherin in U87 cells. In conclusion, miR-124 was found to suppress the migration and invasion of glioma cells in vitro via Capn4.

Topics: 3' Untranslated Regions; Brain Neoplasms; Calpain; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; In Vitro Techniques; MicroRNAs; Neoplasm Invasiveness

Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations.. To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis.. The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA-based silencing of endogenous CAST in cultured ECs facilitated μ-calpain-induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6-driven STAT3 signals. Interleukin-6-induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects.. Loss of CAST in angiogenic ECs facilitates μ-calpain-induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Aorta; Calcium-Binding Proteins; Calpain; Carcinoma, Lewis Lung; Cells, Cultured; Cytokines; Endothelial Cells; Female; Glioblastoma; Humans; Janus Kinases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasms; Neovascularization, Pathologic; Recombinant Fusion Proteins; Retinopathy of Prematurity; Signal Transduction; STAT Transcription Factors; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Vascular Endothelial Growth Factor C

Cleavage of the cell-cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a "protease storm" occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell-cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the "protease storm" could promote the migration and invasion of tumor cells.

Topics: ADAM Proteins; Brain Neoplasms; Calpain; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Furin; Glioblastoma; Glycosylation; Humans; Phosphorylation; Receptor-Like Protein Tyrosine Phosphatases, Class 2

Glioblastoma is an aggressive primary brain tumor with a 5-year survival rate of less than 5%. The ability of glioblastoma cells to invade surrounding brain tissue presents the primary challenge for the success of focal therapeutic approaches. We previously reported that the calcium-activated protease calpain 2 is critical for glioblastoma cell invasion in vitro. Here, we show that expression of calpain 2 is required for the dispersal of glioblastoma cells in a living brain microenvironment. Knockdown of calpain 2 resulted in a 2.9-fold decrease in the invasion of human glioblastoma cells in zebrafish brain. Control cells diffusely migrated up to 450 μm from the site of injection, whereas knockdown cells remained confined in clusters. The invasion study was repeated in organotypic mouse brain tissues, and calpain 2 knockdown cells demonstrated a 2.3-fold lower area of dispersal compared with control cells. In zebrafish brain, glioblastoma cells appeared to migrate in part along the blood vessels of the host. Furthermore, angiogenesis was detected in 27% of zebrafish injected with control cells, whereas only 12.5% of fish receiving knockdown cells showed the formation of new vessels, suggesting a role for calpain 2 in tumor cell angiogenesis. Consistent with the progression of glioblastoma in humans, transplanted tumor cells were not observed to metastasize outside the brain of zebrafish. This study demonstrates that calpain 2 expression is required for the dispersal of glioblastoma cells within the dynamic microenvironment of the brain, identifying zebrafish as a valuable orthotopic system for studying glioblastoma cell invasion.

Topics: Animals; Animals, Genetically Modified; Brain Neoplasms; Calpain; Cell Line, Tumor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Green Fluorescent Proteins; Humans; In Vitro Techniques; Neoplasm Transplantation; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Protein c-fli-1; RNA, Small Interfering; Tumor Cells, Cultured; Zebrafish

Invasion of glioblastoma cells significantly reduces the effectiveness of current treatments, highlighting the importance of understanding dispersal mechanisms and characteristics of the invasive population. Induction of calcium fluxes into glioblastoma cells by autocrine glutamate is critical for invasion. However, the target(s) by which calcium acts to stimulate the dispersal of glioblastoma cells is not clear. In this study, we tested the hypothesis that the calcium-activated protease calpain 2 is required for glioblastoma cell invasion. Knockdown of calpain 2 expression using shRNA or chemical inhibition of calpain activity reduced glioblastoma cell invasion by 90%. Interestingly, decreased expression of calpain 2 did not influence morphology or migration, suggesting regulation of invasion specific mechanisms. Consistent with this idea, 39% less extracellular MMP2 was measured from knockdown cells identifying one mechanism by which calpain 2 mediates glioblastoma cell invasion. This is the first report demonstrating that calpain 2 is required for glioblastoma cell invasion.

Topics: Calpain; Cell Movement; Gene Knockdown Techniques; Glioblastoma; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; RNA, Small Interfering

Glioblastoma grows aggressively due to its ability to maintain abnormally high potentials for cell proliferation. The present study examines the synergistic actions of N-(4-hydroxyphenyl) retinamide (4-HPR) and paclitaxel (PTX) to control the growth of rat glioblastoma C6 and RG2 cell lines. 4-HPR induced astrocytic differentiation that was accompanied by increased expression of the tight junction protein e-cadherin and sustained down regulation of Id2 (member of inhibitor of differentiation family), catalytic subunit of rat telomerase reverse transcriptase (rTERT), and proliferating cell nuclear antigen (PCNA). Flow cytometric analysis showed that the microtubule stabilizer PTX caused cell cycle deregulation due to G2/M arrest. This in turn could alter the fate of kinetochore-spindle tube dynamics thereby halting cell cycle progression. An interesting observation was the induction of G1/S arrest by a combination of 4-HPR and PTX, altering the G2/M arrest induced by PTX alone. This was further ratified by the upregulation of tumor suppressor protein retinoblastoma, which repressed the expression of the key signaling moieties to induce G1/S arrest. Collectively, the combination of 4-HPR and PTX diminished the survival factors (e.g., rTERT, PCNA, and Bcl-2) to make glioblastoma cells highly prone to apoptosis with activation of cysteine proteases (e.g., calpain, cathepsins, caspase-8, caspase-3). Hence, the combination of 4-HPR and PTX can be considered as an effective therapeutic strategy for controlling the growth of heterogeneous glioblastoma cell populations.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Astrocytes; Cadherins; Calpain; Cathepsins; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Survival; Fenretinide; Gene Expression; Glioblastoma; Inhibitor of Differentiation Protein 2; Paclitaxel; Proliferating Cell Nuclear Antigen; Rats; Telomerase; Tumor Suppressor Proteins

Dehydroeburicoic acid (DeEA) is a triterpene purified from medicinal fungi such as Antrodia camphorate, the crude extract of which is known to exert cytotoxic effects against several types of cancer cells. We aim to test the hypothesis that DeEA possesses significant cytotoxic effects against glioblastomas, one of the most frequent and malignant brain tumors in adults. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays indicated that DeEA inhibited the proliferation of the human glioblastoma cell U87MG. In addition, Annexin V and propidium iodide staining showed that DeEA treatment led to a rapid increase of glioblastomas in the necrotic/late apoptotic fraction, whereas cell cycle analysis revealed that DeEA failed to significantly enhance the population of U87MG cells in the hypodiploid (sub-G1) fraction. Using electron microscopy, we found that DeEA induced significant cell enlargements, massive cytoplasmic vacuolization, and loss of mitochondrial membrane integrity. DeEA treatment triggered an intracellular Ca(2+) increase, and DeEA-induced cell death was significantly attenuated by BAPTA-AM but not ethylenediaminetetraacetic acid or ethylene glycol tetraacetic acid. DeEA instigated a reduction of both mitochondrial transmembrane potential and intracellular ATP level. Moreover, DeEA induced proteolysis of alpha-spectrin by calpain, and DeEA cytotoxicity in U87MG cells was caspase-independent but was effectively blocked by calpain inhibitor. Interestingly, DeEA also caused autophagic response that was prevented by calpain inhibitor. Taken together, these results suggest that in human glioblastomas, DeEA induces necrotic cell death that involves Ca(2+) overload, mitochondrial dysfunction, and calpain activation.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Calpain; Caspases; Cell Line, Tumor; Glioblastoma; Humans; Lactate Dehydrogenases; Lanosterol; Membrane Potential, Mitochondrial; Necrosis; Triterpenes

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

Acetazolomide (ACZ) and dexamethasone (DXM) alleviate vasogenic edema and inflammation in glioblastoma patients. Temozolomide (TMZ) is used for treating glioblastoma. We compared modulatory effects of ACZ and DXM on TMZ mediated apoptosis in human glioblastoma T98G and U87MG cells. Cells were treated with drug(s) for 6 h and then left in drug-free medium for 48 h. Although ACZ or DXM alone did not induce apoptosis, TMZ alone induced significant amount of apoptosis. Interestingly, ACZ pretreatment enhanced apoptosis while DXM pretreatment decreased apoptosis. These results suggest that combination chemotherapy with ACZ and TMZ may control inflammation and enhance apoptosis in glioblastoma.

Topics: Acetazolamide; Apoptosis; Aquaporin 1; Brain Edema; Brain Neoplasms; Calpain; Carbonic Anhydrases; Caspases; Cell Line, Tumor; Dacarbazine; Dexamethasone; Drug Interactions; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Inflammation; Mitochondria; Neoplasm Proteins; NF-kappa B; RNA, Messenger; RNA, Neoplasm; Temozolomide; Vascular Endothelial Growth Factor A

Glioblastoma is the most common astrocytic brain tumor in humans. Current therapies for this malignancy are mostly ineffective. Photodynamic therapy (PDT), an exciting treatment strategy based on activation of a photosensitizer, has not yet been extensively explored for treating glioblastoma. We used 5-aminolevulinic acid (5-ALA) as a photosensitizer for PDT to induce apoptosis in human malignant glioblastoma U87MG cells and to understand the underlying molecular mechanisms. Trypan blue dye exclusion test showed a decrease in cell viability after exposure to increasing doses of 5-ALA for 4h followed by PDT with a broad spectrum blue light (400-550 nm) at a dose of 18J/cm(2) for 1h and then incubation at 37 degrees C for 4h. Following 0.5 and 1mM 5-ALA-based PDT (5-ALA-PDT), Wright staining and ApopTag assay showed occurrence of apoptosis morphologically and biochemically, respectively. After 5-ALA-PDT, down regulation of nuclear factor kappa B (NFkappaB) and baculovirus inhibitor-of-apoptosis repeat containing-3 (BIRC-3) protein indicated inhibition of survival signals. Besides, 5-ALA-PDT caused increase in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Activation of calpain, caspase-9, and caspase-3 occurred in course of apoptosis. Calpain and caspase-3 activities cleaved alpha-spectrin at specific sites generating 145kD spectrin breakdown product (SBDP) and 120kD SBDP, respectively. The results suggested that 5-ALA-PDT induced apoptosis in U87MG cells by suppression of survival signals and activation of proteolytic pathways. Thus, 5-ALA-PDT can be an effective strategy for inducing apoptosis in glioblastoma.

Topics: Aminolevulinic Acid; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Baculoviral IAP Repeat-Containing 3 Protein; Brain Neoplasms; Calpain; Caspases; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; NF-kappa B; Peptide Hydrolases; Photochemotherapy; Ubiquitin-Protein Ligases

Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G(2)/M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G(2)/M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50(cdc37), an Hsp90 co-chaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.

Topics: Brain Neoplasms; Calpain; CDC2-CDC28 Kinases; cdc25 Phosphatases; Cell Division; Cell Line, Tumor; Down-Regulation; G2 Phase; Glioblastoma; HSP90 Heat-Shock Proteins; Humans; Rifabutin

Glioblastoma patients receive anti-inflammatory agent for alleviation of vasogenic edema and pain prior to surgery, radiotherapy, and chemotherapy. Oxidative stress is an important mechanism of action of some chemotherapeutic agents in the treatment of glioblastoma. So, we examined the modulatory effects of methylprednisolone (MP, a steroidal anti-inflammatory agent) and indomethacin (IM, a non-steroidal anti-inflammatory agent) on apoptosis in rat C6 glioblastoma cells following oxidative stress with hydrogen peroxide (H(2)O(2)). Exposure of C6 cells to 1 mM H(2)O(2) for 24 h caused significant amounts of morphological and biochemical features of apoptosis. Expressions of Bax and Bcl-2 at mRNA and protein levels were altered resulting in an increase in Bax : Bcl-2 ratio in apoptotic cells, which also exhibited overexpression of 80 kDa calpain and an increase in calpain-cleaved 145 kDa alpha-spectrin breakdown product. Immunofluorescent and propidium iodide labeling detected caspase-3-p20 fragment in apoptotic cells, indicating activation of caspase-3 as well. Treatment of cells with 1 microM MP or 10 microM IM alone did not induce apoptosis. Pretreatment (1 h) with either 1 microM MP or 10 microM IM significantly inhibited H(2)O(2) mediated apoptosis in C6 cells. Thus, pretreatment of glioblastoma with an anti-inflammatory agent, either steroidal or non-steroidal, may compromise the action of a chemotherapeutic agent that mediates therapeutic action via oxidative stress.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Caspase 3; Glioblastoma; Indomethacin; Methylprednisolone; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured

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

Human glioblastoma is a deadly brain tumor that is often treated with a combination of drugs. A new alkylating agent, temozolomide (TMZ), has recently been found efficacious in the clinical trials for glioblastoma. Steroids, such as dexamethasone (DXM), are often used concomitantly as a supportive therapy to treat cerebral edema. However, any possible modulatory effect of the steroids on the efficacy of TMZ has not yet been evaluated experimentally. In this study, we have examined whether DXM provides synergistic or antagonistic effect on TMZ-induced apoptosis in human glioblastoma T98G cells. T98G cells were pretreated with various doses of DXM followed by TMZ. The cell viability was assessed by the trypan blue dye exclusion test. Wright staining and the TdT-mediated dUTP nick-end labeling (TUNEL) assay were used to evaluate apoptotic cell death based on the morphological and biochemical (DNA fragmentation) features, respectively. More biochemical features of apoptotic death, such as upregulation of Bax:Bcl-2 ratio, calpain activity, and caspase-3 activity, were assessed by Western blot analysis. A significant number of T98G cells committed apoptosis after treatment with 200 microM TMZ. However, a pretreatment with 100 microM or 200 microM DXM protected T98G cells against TMZ-induced apoptosis, concomitantly decreasing Bax:Bcl-2 ratio, calpain activity, and caspase-3 activity. These experimental results indicate that DXM works as an antagonistic agent in combination with TMZ. Therefore, our investigation strongly implies that the combination of DXM and TMZ may be counteractive in treating human glioblastoma.

Topics: Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Blotting, Western; Calpain; Cell Line, Tumor; Cell Survival; Dacarbazine; Dexamethasone; DNA Fragmentation; Enzyme Activation; Glioblastoma; Humans; In Situ Nick-End Labeling; Temozolomide; Trypan Blue

Glioblastoma is the deadliest and most prevalent brain tumor. Dexamethasone (DXM) is a commonly used steroid for treating glioblastoma patients for alleviation of vasogenic edema and pain prior to treatment with chemotherapeutic drugs. Temozolomide (TMZ), an alkylating agent, has recently been introduced in clinical trials for treating glioblastoma. Here, we evaluated the modulatory effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells.. Freshly grown cells were treated with different doses of DXM or TMZ for 6 h followed by incubation in a drug-free medium for 48 h. Wright staining and ApopTag assay showed no apoptosis in cells treated with 40 microM DXM but considerable amounts of apoptosis in cells treated with 100 microM TMZ. Apoptosis in TMZ treated cells was associated with an increase in intracellular free [Ca2+], as determined by fura-2 assay. Western blot analyses showed alternations in the levels of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) proteins resulting in increased Bax:Bcl-2 ratio in TMZ treated cells. Western blot analyses also detected overexpression of calpain and caspase-3, which cleaved 270 kD alpha-spectrin at specific sites for generation of 145 and 120 kD spectrin break down products (SBDPs), respectively. However, 1-h pretreatment of cells with 40 microM DXM dramatically decreased TMZ induced apoptosis, decreasing Bax:Bcl-2 ratio and SBDPs.. Our results revealed an antagonistic effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells, implying that treatment of glioblastoma patients with DXM prior to chemotherapy with TMZ might result in an undesirable clinical outcome.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Agents, Hormonal; Apoptosis; bcl-2-Associated X Protein; Brain Neoplasms; Calcium; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Dacarbazine; Dexamethasone; Glioblastoma; Humans; Proto-Oncogene Proteins c-bcl-2; Spectrin; Temozolomide

Cell death in the core of human brain tumors is triggered by hypoxia and lack of nutrients, but the mode of cell death whether necrosis or apoptosis is not clearly defined. To identify the role of apoptosis in brain tumor cell death, we investigated macromolecular (RNA and protein) synthesis and activity in the central to peripheral region of benign [desmoplastic infantile ganglioglioma (DIG) and transitional meningioma (TMG)] and malignant [ependymoma (END), anaplastic astrocytoma (APA), and glioblastoma multiforme (GBM)] brain tumors derived from five patients who had not received previously radiotherapy or chemotherapy. Normal brain tissue (NBT) served as control. RT-PCR analysis of tumor tissues covering central to peripheral regions detected mRNA overexpression of pro-apoptotic gene bax in malignant tumors, indicating a commitment to apoptosis. The mRNA expression of calpain (a Ca(2+)-dependent cysteine protease) and calpastatin (endogenous calpain inhibitor) was altered resulting in an elevated calpain/calpastatin ratio. Calpain content and activity were increased, suggesting a role for calpain in cell death. In the mitochondria-dependent death pathway, caspase-9 and caspase-3 were also overexpressed in tumors. The increased caspase-3 activity cleaved poly(ADP-ribose) polymerase (PARP). Agarose gel electrophoresis detected a mixture of random and internucleosomal DNA fragmentation in malignant brain tumors. Overexpression of pro-apoptotic bax, upregulation of calpain and caspase-3, and occurrence of internucleosomal DNA fragmentation are now presented indicating that one mechanism of cell death in malignant brain tumors is apoptosis, and that enhancement of this process therapeutically may promote decreased tumor growth.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Brain Neoplasms; Calcium-Binding Proteins; Calpain; Caspase 3; Caspase 9; Caspases; DNA Fragmentation; Electrophoresis, Agar Gel; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation