Surgical treatment for brain metastases: Prognostic factors and survival in 309 patients with regard to patient age.

Brain metastases are the most common intracranial tumors. Overall, the only accepted prognostic factors are patient age and performance status. However, several other factors are considered before surgery. We performed a retrospective analysis of 309 patients who underwent surgical resection of newly diagnosed brain metastases between 1994 and 2004. Univariate survival analysis revealed age, performance status, extracranial metastases, complete resection, radiotherapy and re-craniotomy as prognostic indicators. Multivariate analysis determined that patient age, performance status, extracranial metastases, radiotherapy and re-craniotomy are independent factors of prolonged survival. We statistically estimated the age threshold separating patients with favorable outcomes from those with unfavorable prognoses. Using the Kaplan-Meier analysis this threshold can be set at 65 years. Multivariate analysis of patients >65 years revealed the presence of co-morbidities, the number of brain metastases, post-operative performance status and radiotherapy as independent prognostic factors.

Endoglin expression in metastatic breast cancer cells enhances their invasive phenotype.

Endoglin is a cell-surface adhesion protein as well as a coreceptor for transforming growth factor-beta (TGF-beta). It is located on endothelial and few other cells, but also found on certain tumor cells. Brain metastatic breast tumor cells derived from the MDA-MB-231 cell line heavily express endoglin in contrast to the corresponding parental ones. To clarify whether this determines their invasive phenotype, we compared their biological properties with endoglin-silenced brain-metastatic cells, low-expressing parental cells and these transfected with L- and S-endoglins, isoforms transducing or lacking TGF-beta signals. All L-endoglin-overexpressing cells were characterized by numerous invadopodia where endoglin was preferentially localized. Endoglin-expression resulted in elevated levels of the matrix metalloproteinases (MMP-1 and MMP-19) and downregulation of the plasminogen activator inhibitor-1. In Boyden-chamber and wound-healing assays, endoglin-overexpressing cells showed a considerably higher migration and chemotaxis to TGF-beta. In 3D spheroid confrontation assays between breast tumor cells and TGF-beta-secreting glioma cells, high L-endoglin-expressing cells invaded into the glioma-spheroids whereas low-endoglin-expressing cells dissociated in the culture; invasion was blocked by TGF-beta antibodies. In contrast to parental cells, endoglin-overexpressing cells invaded deeply into mouse brain slices. Thus, endoglin expression on tumor cells enhances their invasive character by formation of invadopodia, extracellular proteolysis, chemotaxis and migration.

Molecular biologic and scintigraphic analyses of somatostatin receptor-negative meningiomas.

UNLABELLED: Somatostatin receptor scintigraphy (SRS) using 111In-octreotide has proven useful in the preoperative discrimination of expansive central nervous system lesions. Meningiomas, generally expressing human somatostatin receptor (hsst) on their surface, were detected with a sensitivity of about 100%. This finding was associated with the assumption that meningiomas lack an intact blood-brain barrier. However, this exclusion procedure became questionable when histologically proven meningiomas in which SRS was negative were reported. Therefore, the aim of this study was to discover why these meningiomas gave negative SRS results. METHODS: Before surgery, 46 patients with 47 meningiomas underwent standard MRI and SRS. Thirty-four of these patients with 35 tumors were also examined by 99mTc-diethylenetriaminepentaacetic acid (DTPA) brain scintigraphy. After surgical resection, hsst subtype 2 (hsst2) messenger RNA (mRNA) expression of 4 SRS-positive and 4 SRS-negative meningiomas was estimated semiquantitatively by reverse transcriptase polymerase chain reaction (RT-PCR). Translation of hsst2 mRNA into receptor proteins was proven immunocytochemically on the surface of 1 SRS-positive and 1 SRS-negative meningioma. Tumor specimens used for RNA extraction and RT-PCR and cultivated cells used for hsst2 immunostaining were tested for their meningioma nature by immunochemistry. RESULTS: SRS yielded positive results in 39 meningiomas with a tumor volume of 24.1 +/- 32.8 mL and negative results in 8 meningiomas with a volume of 3.9 +/- 6.5 mL. 99mTc-DTPA scintigraphy visualized 24 of 35 meningiomas. SRS was positive in all of them. In contrast, 11 meningiomas were (99mTc-DTPA negative. In these meningiomas, SRS was negative in 5 cases (5.4 +/- 8.1 mL), whereas the remaining 6 were positive (4.6 +/- 4.5 mL). None of the meningiomas was 99mTc-DTPA positive and SRS negative. RT-PCR revealed no significant difference of hsst2 mRNA expression between SRS-positive and SRS-negative meningiomas but showed varied expression among all meningiomas regardless of SRS results. Furthermore, hsst2 proteins were visualized immunocytochemically on the surface of cultivated cells of SRS-positive and SRS-negative meningiomas. CONCLUSION: SRS-negative meningiomas do express hsst2; thus, in these meningiomas SRS is false-negative. Because an insufficient sensitivity was excluded, 99mTc-DTPA scintigraphy identified a permeability barrier in SRS-negative meningiomas that explains their false-negative SRS results. SRS-negative meningiomas most likely meet the function of their tissue of origin (the meninges) to develop more-or-less intact permeability barriers.

Detection of natural peptide antibiotics in human nasolacrimal ducts.

PURPOSE: To determine the expression and production of antimicrobial peptides by mucosal cells of the lacrimal passage in healthy and pathologic states. METHODS: Detection of bactericidal-permeability-increasing protein (BPI), heparin-binding protein (CAP37), human cationic antimicrobial protein (LL-37), human alpha-defensin 5 (HD5), human alpha-defensin 6 (HD6), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was performed by reverse transcription-polymerase chain reaction (RT-PCR). Intracellular deposition of lysozyme, lactoferrin, secretory phospholipase A(2), human neutrophil defensins (HNP-1, -2, and -3), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was analyzed immunohistochemically. Samples were obtained from 15 patients by surgery and from 10 cadavers. RESULTS: RT-PCR revealed BPI, CAP37, and HBD-1 mRNA in samples of healthy nasolacrimal duct epithelium. Additionally, HBD-2 mRNA was detected in epithelial samples from patients with dacryocystitis. Messenger RNAs for LL-37 and alpha-defensin 5 and 6 were absent in all samples investigated. Immunohistochemistry revealed lysozyme, lactoferrin, secretory phospholipase A(2), and HNP-1, -2, and -3 to be present in all samples, whereas HBD-1 was present only in some of the healthy and inflamed samples. Immunoreactive HBD-2 peptide was visible only in some of the inflamed samples. CONCLUSIONS: The data suggest that the human efferent tear ducts produce a broad spectrum of antimicrobial peptides. Under inflammatory conditions, changes in the expression pattern occurred, revealing induction of the human inducible defensin HBD-2 and in some cases downregulation of HBD-1 and CAP37. Antimicrobial peptides have a therapeutic potential in dacryocystitis, in that they have a broad spectrum of antimicrobial activity and accelerate epithelial healing. However, caution is appropriate, because defensins also promote fibrin formation and cell proliferation, which are key elements in scarring processes, such as dacryostenosis.

ATP and adenosine induce ramification of microglia in vitro.

Microglial cells in the healthy adult brain possess a characteristic ramified morphology with multiple branched processes, small somata and down-regulated inflammatory properties. In contrast, microglial cells isolated from new-born rat brain inevitably show a non-ramified amoeboid phenotype, which is observed in vivo after pathologic activation or during development. To identify factors that control microglial morphology we investigated the effects of purines alone or in combination with astrocyte-conditioned medium (ACM). Under optimized culture conditions postnatal rat microglial cells developed an amoeboid to ovoid phenotype. Addition of 0.6-1 mM ATP or adenosine induced the outgrowth of numerous processes after 2-3 days that could be observed also in the presence of ACM as previously reported. Culture in ACM plus ATP or adenosine yielded an optimized ramified phenotype. ATP or adenosine, but not ACM alone, also prevented the formation of a flat, amoeboid morphology induced by lipopolysaccharide (LPS); however, at 0.6-1 mM they did not reduce the initial LPS-induced activation of the transcription factor NF-kappaB. By using specific agonists or antagonists the morphological transformations could not be confined to a distinct purinoreceptor subtype, but appeared to be mediated by long-term presence of adenosine in the medium to which phosphorylated purines were rapidly hydrolyzed by microglial cells. Since ACM did not contain sufficient concentrations of ATP or adenosine, purines are not the only ramification-inducing factors present in ACM; however, they are a valuable tool to induce microglial ramification in vitro.

Somatostatin inhibits the production of vascular endothelial growth factor in human glioma cells.

In various cell types, the neuro- and endocrine peptide somatostatin induces inhibitory and anti-secretory effects. Since somatostatin receptors, especially of the subtype sst2A, are constantly over-expressed in gliomas, we investigated the influence of somatostatin and the receptor subtype-selective peptide/non-peptide agonists octreotide and L-054,522 on the secretion of the most important angiogenesis factor produced by gliomas, vascular endothelial growth factor (VEGF). Cultivated cells from solid human gliomas of different stages and glioma cell lines secreted variable amounts of VEGF, which could be lowered to 25% to 80% by co-incubation with somatostatin or sst2-selective agonists (octreotide and L-054,522). These effects were dose-dependent at nanomolar concentrations. Stimulation with different growth factors (EGF, bFGF) or hypoxia considerably increased VEGF production over basal levels. Growth factor-induced VEGF synthesis could be suppressed to <50% by co-incubation with somatostatin or an sst2-selective agonist; this was less pronounced in hypoxia-induced VEGF synthesis. The effects were detected at the protein and mRNA levels. These experiments indicate a potent anti-secretory action of somatostatin or sst2 agonists on human glioma cells that may be useful for inhibiting angiogenesis in these tumors.

Topology of the signal transduction of the G protein-coupled somatostatin receptor sst2 in human glioma cells.

By a dual approach, using electron microscopy and biochemical techniques, we investigated the topology of the somatostatin receptor sst2 with its inhibitory G protein Gialpha after ligand-induced stimulation and internalization in human glioma cells. On intact cells, the sst2 was labeled at 8 degrees C by an antibody directed to its extracellular sequence followed by a 15-nm gold-labeled secondary antibody. In the presence of the ligand, internalization was induced by exposure to 37 degrees C for 5-10 min. Then, cells were either fixed for immunoelectron-microscopic analysis or homogenized for density gradient separation. After post-embedding staining of the sst2-labeled sections with anti-Gialpha1- 3 or anti-caveolin, a co-localization of sst2, Gialpha and caveolin was detected in endosomal vesicles after 5 min of internalization, but not after 10 min. Furthermore, the gold-labeled organelles containing the internalised receptor were separated from the non-labeled ones on sucrose gradients (density shift separation) and analyzed by Western blotting. Also here, in fractions with higher densities, sst2 could be costained with Gialpha and caveolin after 5 min. From these congruent results from both methods, it can be concluded that, in human glioma cells, the receptor sst2 (1) is internalised in caveolin-positive vesicles and (2) is neighboured to its Gialpha proteins at the plasma membrane and early endosomes.

Influence of the somatostatin receptor sst2 on growth factor signal cascades in human glioma cells.

The somatostatin receptor subtype sst2A is highly expressed, non-mutated and functionally active in gliomas. After stimulation of cultivated human U343 glioma cells with somatostatin, octreotide (sst2-, sst3- and sst5-selective peptide agonist) or the sst2-selective non-peptide agonist L-054,522 multiple signal transduction pathways are induced: elevated cAMP levels are reduced, protein tyrosine phosphatases (especially SHP2) are activated and mitogen-activated protein kinases are inhibited. Stimulation of the phosphatases resulted in dephosphorylation of activated receptors for EGF and PDGF (epidermal and platelet-derived growth factor), and as a consequence the mitogen-activated protein kinases ERK 1 and 2 (p42/p44) were de-phosphorylated in co-stimulation experiments. Furthermore, somatostatin or sst2-selective agonists reduced EGF-stimulated expression of the AP-1 complex (c-jun/c-jun) on the transcriptional and translational level. These experiments show that the interaction of stimulatory and inhibitory receptors are important mechanisms for the regulation of signal cascades and gene expression.

Somatostatin receptors in gliomas.

Gliomas differ from non-malignant glial cells in the overexpression or mutations of genes involved in cell cycle or growth regulation. One example is the overexpression of the somatostatin receptor subtype 2 (sst2), especially of the splice variant sst2A. The reasons for this overexpression are not known. However, the coding sequence and part of the promoter region is not mutated. In accordance to this, the sst2 is functionally active and is internalised upon agonist stimulation. Immunoelectronmicroscopic studies show that the activated sst2 is internalised via caveolin-positive endosomal vesicles and later accumulates in multivesicular bodies and lysosomal compartments. The activated sst2 is found to be co-localised with the inhibitory G-protein Gialpha at the plasma membrane and in early endosomal vesicles. Multiple signal transduction pathways are induced. Stimulation of sst2 lowers cAMP levels elicited by forskolin and activates the protein tyrosine phosphatase SHP-2. In contrast to other sst2-expressing cells a long term antiproliferative effect of somatostatin or sst2-selective agonists are not detected in cultivated glioma cells. However, continuous stimulation of sst2 decreases the expression of genes promoting tumour survival.

Molecular analysis of the somatostatin receptor subtype 2 in human glioma cells.

Gliomas constantly overexpress the receptor subtype SST2 for the inhibitory peptide somatostatin. Since somatostatin or metabolically stable agonists like octreotide have an antiproliferative and antisecretory potential for the treatment of SST2-expressing tumors, we evaluated the molecular integrity of SST2 in gliomas on the DNA, mRNA and protein levels. Sequencing of about 1800 bases from the SST2 gene in nine gliomas and five control samples revealed no mutations, but polymorphisms were detected in the 5'-region irrespective of the malignancy of the sample. Gliomas and the human glioma cell line U343 expressed mRNA for the receptor splice variant SST2A with a size of about 4.2 kb. A novel antibody generated against an extracellular part of the SST2 amino acid sequence strongly reacted with an 75-kDa protein in membranes from glioma or meningioma cells and-much weaker-normal rat astrocytes. The receptor could be immunostained on the surface of intact glioma cells or (weaker) astrocytes at the light and electron microscopic level. These results show that the somatostatin receptor SST2 is non-mutated in gliomas and has similar molecular properties as in non-malignant cells.