Search

PDF file - 65K, Effect of temsirolimus in a subcutaneous xenograft mouse model

Purpose: Meningiomas are frequent intracranial or spinal neoplasms, which recur frequently and can show aggressive clinical behaviour. We elucidated the impact of the integrin inhibitor cilengitide on migration, proliferation, and radiosensitization of meningioma cells.Experimental Design: We analyzed integrin expression in tissue microarrays of human meningiomas and the antimeningioma properties of cilengitide in cell cultures, subcutaneous and intracranial nude mouse models by measuring tumor volumes and survival times.Results: αvβ5 was the predominantly expressed integrin heterodimer in meningiomas, whereas αvβ3 was mainly detected in tumor blood vessels. Application of up to 100 μg/mL cilengitide resulted in only mildly reduced proliferation/survival of meningioma cell lines. Effects on cell survival could be enhanced by irradiation. One μg/mL cilengitide was sufficient to significantly inhibit meningioma cell migration and invasion in vitro. A daily dosage of 75 mg/kg did neither affect tumor volumes nor overall survival (P = 0.813, log-rank test), but suppressed brain invasion in a significant fraction of treated animals. A combination of 75 mg/kg cilengitide daily and irradiation (2 × 5 Gy) led to a 67% reduction of MRI-estimated tumor volumes in the intracranial model (P < 0.01), whereas the corresponding reduction reached by irradiation alone was only 55% (P < 0.05).Conclusions: These data show that a monotherapy with cilengitide is not likely to achieve major responses in rapidly growing malignant meningiomas, although brain invasion may be reduced because of the strong antimigratory properties of the drug. The combination with radiotherapy warrants further attention. Clin Cancer Res; 19(19); 5402–12. ©2013 AACR.

Supplementary Figure 1 - PDF file 57K, integrin mRNA expression in meningiomas

PDF file - 69K, Real time-PCR primer sequences

Supplementary Figure 3 - PDF file 43K, Overall survival of mice treated with cilengitide and control animals

Supplementary Figure 2 - PDF file 128K, Cell cycle analyses under cilengitide treatment. Meningioma cell lines treated with cilengitide and analysed for cell cycle changes

Purpose: To evaluate the mTORC1 (mammalian target of rapamycin complex 1) pathway in meningiomas and to explore mTORC1 as a therapeutic target in meningioma cell lines and mouse models.Experimental Design: Tissue microarrays (53 meningiomas of all WHO grades) were stained for phosphorylated polypeptides of mTOR, Akt, and the mTORC1 targets 4EBP1 and p70S6K, the latter being the consensus marker for mTORC1 activity. Expression of proteins and mRNAs was assessed by Western blotting and real-time PCR in 25 tumors. Cell lines Ben-Men-1 (benign), IOMM-Lee and KT21 (malignant), and pairs of merlin-positive or -negative meningioma cells were used to assess sensitivity toward mTORC1 inhibitors in methyl-tetrazolium and bromodeoxyuridine (BrdUrd) assays. The effect of temsirolimus (20 mg/kg daily) on tumor weight or MRI-estimated tumor volume was tested by treatment of eight nude mice (vs. 7 controls) carrying subcutaneous IOMM-Lee xenografts, or of eight (5) mice xenotransplanted intracranially with IOMM-Lee (KT21) cells in comparison to eight (5) untreated controls.Results: All components of the mTORC1 pathway were expressed and activated in meningiomas, independent of their WHO grade. A significant dosage-dependent growth inhibition by temsirolimus and everolimus was observed in all cell lines. It was slightly diminished by merlin loss. In the orthotopic and subcutaneous xenograft models, temsirolimus treatment resulted in about 70% growth reduction of tumors (P < 0.01), which was paralleled by reduction of Ki67 mitotic index (P < 0.05) and reduction of mTORC1 activity (p70S6K phosphorylation) within the tumors.Conclusion: mTORC1 inhibitors suppress meningioma growth in mouse models, although the present study did not measure survival. Clin Cancer Res; 19(5); 1180–9. ©2012 AACR.

Deep perforator arteriopathy (DPA) and cerebral amyloid angiopathy (CAA) are the commonest known cerebral small vessel diseases (CSVD), which cause ischaemic stroke, intracebral haemorrhage (ICH) and vascular cognitive impairment (VCI). While thus far mainly considered as separate entities, we here propose that DPA and CAA share similarities, overlap and interact, so that ‘pure’ DPA or CAA are extremes along a continuum of age-related small vessel pathologies. We suggest blood-brain barrier (BBB) breakdown, endothelial damage and impaired perivascular β-amyloid (Aβ) drainage are hallmark common mechanisms connecting DPA and CAA. We also suggest a need for new biomarkers (e.g. high-resolution imaging) to deepen understanding of the complex relationships between DPA and CAA.

Deep perforator arteriopathy (DPA) and cerebral amyloid angiopathy (CAA) are the commonest known cerebral small vessel diseases (CSVD), which cause ischaemic stroke, intracebral haemorrhage (ICH) and vascular cognitive impairment (VCI). While thus far mainly considered as separate entities, we here propose that DPA and CAA share similarities, overlap and interact, so that 'pure' DPA or CAA are extremes along a continuum of age-related small vessel pathologies. We suggest blood-brain barrier (BBB) breakdown, endothelial damage and impaired perivascular β-amyloid (Aβ) drainage are hallmark common mechanisms connecting DPA and CAA. We also suggest a need for new biomarkers (e.g. high-resolution imaging) to deepen understanding of the complex relationships between DPA and CAA.

Mesenchymal stem cells (MSC) have been found in various cancers and were discussed to influence tumor biology. Cells fulfilling the complete MSC criteria, including surface marker expression (CD73, CD90, CD105) and tri-lineage differentiation, have been isolated solely from a low percentage of high-grade meningiomas. In contrast, pure co-expression of the surface-markers was relatively frequent, raising the question for an additional role of these membrane proteins in meningiomas. Therefore, here we analyzed the expression of CD73, CD90 and CD105 in a series of meningiomas of all grades. Although no significant association of any marker with meningeal tumor growth per se or with tumor-grade was observed, we detected a positive Pearson correlation (r = 0.55, p ≤ 0.05) in low-grade tumors between CD73 and the most relevant tumor suppressor NF2/Merlin, supported by a tendency of lower CD73 expression in cases with allelic losses at the NF2-locus, which express significantly lower NF2/Merlin-mRNA (p ≤ 0.05). In two pairs of syngenous meningeal or meningioma cell lines with or without shRNA-mediated knockdown of NF2/Merlin a nearly complete loss of CD73 mRNA expression was observed after the knockdown (p ≤ 0.001). This suggested that the correlation observed in tumors may result from a direct functional link between Merlin and CD73. Since CD73 is a 5′-exonucleotidase (termed NT5E), we discuss a potential role of NT5E-mediated purinergic signaling to modulate actin-cytoskeleton and cell contacts, which may be a functional link to NF2/Merlin.

Benjamin Voellger,1 Elmar Kirches,2 Annette Wilisch-Neumann,2 Andreas Weise,1 Jorge Humberto Tapia-Perez,1 Rosita Rupa,1 Christian Mawrin,2 Raimund Firsching11Department of Neurosurgery, 2Department of Neuropathology, Otto von Guericke University, Magdeburg, GermanyObjective: Resveratrol is a phytoestrogen with various antiproliferative and proapoptotic effects. This in vitro study aimed to analyze the effect of resveratrol on the viability and expression of modulators of apoptosis in GH3 pituitary adenoma cells of the rat.Methods: GH3 cells were incubated with resveratrol concentrations from 20 to 100 µM for 48–72 hours. Cell viability was quantified using a hemocytometer. We assessed the ability of resveratrol to kill GH3 cells by an enzyme-linked immunosorbent assay (ELISA) of nucleosome liberation and by DNA degradation (unidimensional gel electrophoresis). Relative messenger RNA (mRNA) expression of survivin, B-cell lymphoma-2 protein (BCL-2) and BCL-2-associated X protein (BAX) normalized to β2 microglobulin was measured using quantitative real-time polymerase chain reaction (qRT-PCR).Results: GH3 cell survival significantly decreased with increasing concentrations of resveratrol. In GH3 cells treated with 100 µM resveratrol, ELISA demonstrated a significant rise of nucleosome liberation, which typically occurs during apoptosis. In parallel, gel electrophoresis showed degradation of DNA into random fragments, pointing to a necrotic mode of cell death in most GH3 cells. In GH3 cells treated with 100 µM resveratrol, qRT-PCR detected a significant decrease of BCL-2 mRNA expression and a decrease of survivin mRNA expression, whereas a change of BAX mRNA expression could not be found. The BAX/BCL-2 ratio was significantly increased in GH3 cells after resveratrol treatment.Conclusions: Resveratrol reduces GH3 cell viability in a dose-dependent manner by inducing nonapoptotic cell death and apoptosis. Apoptosis in GH3 cells is probably mediated by resveratrol-dependent downregulation of apoptosis inhibitors, namely BCL-2 and possibly survivin. Further investigation of the potential effects of resveratrol on pituitary adenoma cells is warranted.Keywords: resveratrol, phytoestrogens, viability, GH3 cells

Purpose: To evaluate the mTORC1 (mammalian target of rapamycin complex 1) pathway in meningiomas and to explore mTORC1 as a therapeutic target in meningioma cell lines and mouse models. Experimental Design: Tissue microarrays (53 meningiomas of all WHO grades) were stained for phosphorylated polypeptides of mTOR, Akt, and the mTORC1 targets 4EBP1 and p70S6K, the latter being the consensus marker for mTORC1 activity. Expression of proteins and mRNAs was assessed by Western blotting and real-time PCR in 25 tumors. Cell lines Ben-Men-1 (benign), IOMM-Lee and KT21 (malignant), and pairs of merlin-positive or -negative meningioma cells were used to assess sensitivity toward mTORC1 inhibitors in methyl-tetrazolium and bromodeoxyuridine (BrdUrd) assays. The effect of temsirolimus (20 mg/kg daily) on tumor weight or MRI-estimated tumor volume was tested by treatment of eight nude mice (vs. 7 controls) carrying subcutaneous IOMM-Lee xenografts, or of eight (5) mice xenotransplanted intracranially with IOMM-Lee (KT21) cells in comparison to eight (5) untreated controls. Results: All components of the mTORC1 pathway were expressed and activated in meningiomas, independent of their WHO grade. A significant dosage-dependent growth inhibition by temsirolimus and everolimus was observed in all cell lines. It was slightly diminished by merlin loss. In the orthotopic and subcutaneous xenograft models, temsirolimus treatment resulted in about 70% growth reduction of tumors (P < 0.01), which was paralleled by reduction of Ki67 mitotic index (P < 0.05) and reduction of mTORC1 activity (p70S6K phosphorylation) within the tumors. Conclusion: mTORC1 inhibitors suppress meningioma growth in mouse models, although the present study did not measure survival. Clin Cancer Res; 19(5); 1180–9. ©2012 AACR.

Objective: Statins are cholesterol reducers with a considerable dose-dependent anti-tumoral effect. The apoptotic effect could be increased by combining statins or by adding pioglitazone (PGZ). The last one is an anti-diabetic drug, an agonist of PPAR-y receptor. We have demonstrated the efficacy of[for full text, please go to the a.m. URL], 65. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)

Purpose: Meningiomas are frequent intracranial or spinal neoplasms, which recur frequently and can show aggressive clinical behaviour. We elucidated the impact of the integrin inhibitor cilengitide on migration, proliferation, and radiosensitization of meningioma cells. Experimental Design: We analyzed integrin expression in tissue microarrays of human meningiomas and the antimeningioma properties of cilengitide in cell cultures, subcutaneous and intracranial nude mouse models by measuring tumor volumes and survival times. Results: αvβ5 was the predominantly expressed integrin heterodimer in meningiomas, whereas αvβ3 was mainly detected in tumor blood vessels. Application of up to 100 μg/mL cilengitide resulted in only mildly reduced proliferation/survival of meningioma cell lines. Effects on cell survival could be enhanced by irradiation. One μg/mL cilengitide was sufficient to significantly inhibit meningioma cell migration and invasion in vitro. A daily dosage of 75 mg/kg did neither affect tumor volumes nor overall survival (P = 0.813, log-rank test), but suppressed brain invasion in a significant fraction of treated animals. A combination of 75 mg/kg cilengitide daily and irradiation (2 × 5 Gy) led to a 67% reduction of MRI-estimated tumor volumes in the intracranial model (P < 0.01), whereas the corresponding reduction reached by irradiation alone was only 55% (P < 0.05). Conclusions: These data show that a monotherapy with cilengitide is not likely to achieve major responses in rapidly growing malignant meningiomas, although brain invasion may be reduced because of the strong antimigratory properties of the drug. The combination with radiotherapy warrants further attention. Clin Cancer Res; 19(19); 5402–12. ©2013 AACR.

Objective: Resveratrol is a phytoestrogen with various antiproliferatic and proapoptotic effects. One of the underlying mechanisms is downregulation of apoptosis inhibitors, such as Survivin. In human pituitary adenoma cells, Survivin expression is increased as compared to healthy pituitary gland tissue.[for full text, please go to the a.m. URL], 63. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie (JNS)

The mitochondrial protein frataxin (FXN) is known to be involved in mitochondrial iron homeostasis and iron-sulfur cluster biogenesis. It is discussed to modulate function of the electron transport chain and production of reactive oxygen species (ROS). FXN loss in neurons and heart muscle cells causes an autosomal-dominant mitochondrial disorder, Friedreich's ataxia. Recently, tumor induction after targeted FXN deletion in liver and reversal of the tumorigenic phenotype of colonic carcinoma cells following FXN overexpression were described in the literature, suggesting a tumor suppressor function. We hypothesized that a partial reversal of the malignant phenotype of glioma cells should occur after FXN transfection, if the mitochondrial protein has tumor suppressor functions in these brain tumors. In astrocytic brain tumors and tumor cell lines, we observed reduced FXN levels compared with non-neoplastic astrocytes. Mitochondrial content (citrate synthase activity) was not significantly altered in U87MG glioblastoma cells stably overexpressing FXN (U87-FXN). Surprisingly, U87-FXN cells exhibited increased cytoplasmic ROS levels, although mitochondrial ROS release was attenuated by FXN, as expected. Higher cytoplasmic ROS levels corresponded to reduced activities of glutathione peroxidase and catalase, and lower glutathione content. The defect of antioxidative capacity resulted in increased susceptibility of U87-FXN cells against oxidative stress induced by H(2)O(2) or buthionine sulfoximine. These characteristics may explain a higher sensitivity toward staurosporine and alkylating drugs, at least in part. On the other hand, U87-FXN cells exhibited enhanced growth rates in vitro under growth factor-restricted and hypoxic conditions and in vivo using tumor xenografts in nude mice. These data contrast to a general tumor suppressor function of FXN but suggest a dual, pro-proliferative but chemosensitizing role in astrocytic tumors.

The immediate early transcription factor nuclear factor (IκBs) kappa B (NF-κB) is crucially involved in the regulation of numerous physiological or pathophysiological processes such as inflammation and tumourigenesis. Therefore, the control of NF-κB activity, which is mainly regulated by signal-induced degradation of cytoplasmic inhibitors of NF-κB (IκBs), is of high relevance. One known alternative pathway of NF-κB regulation is the stimulus-induced proteasomal degradation of RelB, a component of the NF-κB dimer. Here, we identified the serine/threonine protein kinase glycogen synthase kinase-3β (GSK-3β) as a critical signalling component leading to RelB degradation. In Jurkat leukaemic T cells as well as in primary human T cells, tetradecanoylphorbolacetate/ionomycin- and CD3/CD28-induced RelB degradation were impaired by a GSK-3β-specific pharmacological inhibitor, an ectopically expressed dominant-negative GSK-3β mutant and by small-interfering RNA-mediated silencing of GSK-3β expression. Furthermore, a physical interaction between RelB and GSK-3β was shown by co-immunoprecipitation, which was already notable in unstimulated cells. Most importantly, as demonstrated by in vitro kinase assays, human RelB is inducibly phosphorylated by GSK-3β, indicating a direct substrate-enzyme relationship. The serine residue 552 is a target of GSK-3β-mediated phosphorylation in vitro and in vivo. We conclude that GSK-3β is a crucial regulator of RelB degradation, stressing the relevant linkage between the NF-κB system and GSK-3β.

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

Benjamin Voellger,1 Elmar Kirches,2 Annette Wilisch-Neumann,2 Andreas Weise,1 Jorge Humberto Tapia-Perez,1 Rosita Rupa,1 Christian Mawrin,2 Raimund Firsching11Department of Neurosurgery, 2Department of Neuropathology, Otto von Guericke University, Magdeburg, GermanyObjective: Resveratrol is a phytoestrogen with various antiproliferative and proapoptotic effects. This in vitro study aimed to analyze the effect of resveratrol on the viability and expression of modulators of apoptosis in GH3 pituitary adenoma cells of the rat.Methods: GH3 cells were incubated with resveratrol concentrations from 20 to 100 µM for 48–72 hours. Cell viability was quantified using a hemocytometer. We assessed the ability of resveratrol to kill GH3 cells by an enzyme-linked immunosorbent assay (ELISA) of nucleosome liberation and by DNA degradation (unidimensional gel electrophoresis). Relative messenger RNA (mRNA) expression of survivin, B-cell lymphoma-2 protein (BCL-2) and BCL-2-associated X protein (BAX) normalized to β2 microglobulin was measured using quantitative real-time polymerase chain reaction (qRT-PCR).Results: GH3 cell survival significantly decreased with increasing concentrations of resveratrol. In GH3 cells treated with 100 µM resveratrol, ELISA demonstrated a significant rise of nucleosome liberation, which typically occurs during apoptosis. In parallel, gel electrophoresis showed degradation of DNA into random fragments, pointing to a necrotic mode of cell death in most GH3 cells. In GH3 cells treated with 100 µM resveratrol, qRT-PCR detected a significant decrease of BCL-2 mRNA expression and a decrease of survivin mRNA expression, whereas a change of BAX mRNA expression could not be found. The BAX/BCL-2 ratio was significantly increased in GH3 cells after resveratrol treatment.Conclusions: Resveratrol reduces GH3 cell viability in a dose-dependent manner by inducing nonapoptotic cell death and apoptosis. Apoptosis in GH3 cells is probably mediated by resveratrol-dependent downregulation of apoptosis inhibitors, namely BCL-2 and possibly survivin. Further investigation of the potential effects of resveratrol on pituitary adenoma cells is warranted.Keywords: resveratrol, phytoestrogens, viability, GH3 cells

e22140 Background: Meningiomas are frequent intracranial tumors, which may show high rates of tumor recurrence after surgery. Additionally, a number of meningiomas show aggressive clinical behaviour with significant reduction of patient´s survival. In the present study, the impact of cilengitide, an integrin inhibitor, on migration, proliferation and radiosensitization of meningioma cells was evaluated. Methods: We analyzed integrin expression in tissue microarray samples and the anti-meningioma properties of cilengitide in vitro using different meningioma cell lines including pairs with knockdown of NF2, and tested the substance in subcutaneous and intracranial orthotopic nude mice models. Results: The predominantly expressed integrin in the tumor cells was αvβ5, while αvβ3 was mainly restricted to tumor blood vessels. One µM cilengitide was sufficient to significantly inhibit meningioma cell migration and invasion in vitro. On the other hand, even high doses of the drug resulted in only mildly reduced proliferation/survival of the cells, although the effects on cell survival were improved by irradiation. A daily dosage of up to 75 mg/kg did not affect tumor volumes in both mouse models. However, a combination of 75 mg/kg cilengitide daily and irradiation (2 x 5 Gy) led to a stronger reduction of MRI estimated tumor volumes in the intracranial model (67%, p