calpain and Brain-Neoplasms

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

Several human acute and chronic diseases involve calpain over-activation. However, the mechanistic linkages between the etiology and the progression of cell damages are not yet completely understood. Here we show that different human cells and tissues, including brain tumor specimens, cell lines of nerve origin, breast tumor samples and peripheral blood mononuclear cells from healthy donors, express a calpastatin form that lacks all the exons coding for the domains responsible of calpain inhibition. The open reading frame of this new form of calpastatin, named hcast 3-25, starts inside the L-domain (exons 2 and 3) and continues with the exons from 25 to 29 that code for the conserved C-terminal tail shared by all the full-length calpastatins. We have here observed that unlike the other calpastatins forms, that are predominantly Δ3 splice variants, hcast 3-25 is endowed with exon 3. At a functional level, recombinant hcast 3-25 operates as a positive modulator of calpain-1 in vitro by preventing 1) calpain-1-mediated proteolytic degradation of the activated enzyme and 2) binding to calpain-1 of inhibitory calpastatins that contain the L-domain. Thus hcast 3-25 can be considered as a novel member and possible modulator of the calpain/calpastatin system acting by a mechanism alternative to inhibition.

Topics: Brain Neoplasms; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Humans; Neoplasm Proteins; Protein Domains; Protein Isoforms; 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

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.

Topics: Autophagosomes; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Proteins; Brain Neoplasms; Calpain; Cell Adhesion; Cell Line, Tumor; Chemotaxis; Endocytosis; Glioma; HEK293 Cells; Humans; Phosphatidylinositol 3-Kinases; Receptors, CXCR4; Receptors, G-Protein-Coupled; TOR Serine-Threonine Kinases

Recent evidence indicates that the increased expression of calpain small subunit 1 (Capn4) is associated with tumorigenesis. This study was designed to explore the role which Capn4 plays in human glioma.. We detected the expression of Capn4 by immunohistochemistry in tissue microarrays and tissue samples. Following the down-regulation of Capn4 in glioma cell lines by a specific short hairpin RNA, the function of Capn4 in invasion, migration, and proliferation was assessed. We then evaluated the prognostic role of Capn4 using univariate and multivariate analysis in 94 glioblastoma (GBM) patients.. Glioma tissues exhibited notably higher expression of Capn4 compared with control brain tissues and was positively correlated with histological malignancy. The down-regulation of Capn4 in glioma cells led to a decrease in invasion and migration in vitro. Through univariate analysis, the prognosis of GBM patients with Capn4 overexpression was significantly poorer with respect to progression-free survival (PFS) and overall survival (OS). Based on the results of the multivariate analysis, Capn4(high) was demonstrated to be a negative independent prognostic indicator for PFS and OS in GBM patients.. The overexpression of Capn4 is a novel negative prognostic marker, and Capn4 may be used as a new target in therapeutic strategies for human glioma.

Topics: Adolescent; Adult; Aged; Brain Neoplasms; Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Follow-Up Studies; Glioma; Humans; Male; Middle Aged; Neoplasm Invasiveness; RNA, Small Interfering; Severity of Illness Index; Statistics, Nonparametric; Tissue Array Analysis; Wound Healing; Young Adult

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

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.. This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.. Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain Neoplasms; Calpain; Cell Line, Tumor; Dacarbazine; Glioma; Humans; Male; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Selenium; Skin Neoplasms; Temozolomide; Xenograft Model Antitumor Assays

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 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

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

The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between mu-calpain activation and merlin proteolysis induced by the oxidative stress.. The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H2O2) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses.. Despite the consistent expressions of activated mu-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both mu-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated mu-calpain at the plasma membrane, and, after mu-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus.. These findings suggest that oxidative stress-induced activation of mu-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

Topics: Brain Neoplasms; Calpain; Cell Adhesion; Cell Transformation, Neoplastic; Female; Humans; Hydrogen Peroxide; Male; Meningioma; Middle Aged; Neurofibromin 2; Oxidants; Oxidative Stress; Tumor Cells, Cultured

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

Topics: Base Sequence; Brain Neoplasms; Calpain; Cell Line; DNA Primers; Enzyme Activation; Genes, Neurofibromatosis 2; Glioma; Glutathione Transferase; Humans; Membrane Proteins; Meningioma; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurilemmoma; Neurofibromin 2; Polymerase Chain Reaction; Recombinant Fusion Proteins; RNA, Messenger; Templates, Genetic; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Both ice-like protease and calpain have been shown to be involved in apoptosis in non-neuronal cells. Cultured rat cerebellar granule neurons undergo apoptosis when exposed to low potassium-containing medium. Calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and N-acetyl-Leu-Leu-Met-CHO (calpain inhibitor II) as well as interleukin-beta 1 converting enzyme (ICE)-like protease inhibitor Z-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) protect against such apoptotic death. They also reduce DNA laddering and the number of apoptotic nuclei. Staurosporine treatment also evokes apoptosis in human neuroblastoma SH-SY5Y. While Z-D-DCB is again anti-apoptotic, calpain inhibitors only provide modest effects in this model. Our results suggest that ICE-like protease plays a critical role in neuronal apoptosis whereas the contributions of calpain are more cell-type dependent.

Topics: Animals; Apoptosis; Brain Neoplasms; Calpain; Caspase 1; Coloring Agents; Cysteine Endopeptidases; DNA Fragmentation; Humans; L-Lactate Dehydrogenase; Neuroblastoma; Neurons; Protease Inhibitors; Rats; Tumor Cells, Cultured