Quantitative profiling of genes associated with cancer pathways in brain tumors.

BACKGROUND: Brain tumors are a heterogeneous group of malignancies characterized by inter- and intratumoral heterogeneity. Among them, the most aggressive and, despite advances in medicine, still incurable remains glioblastoma. One of the reasons is the high recurrence rate of the disease and resistance to temozolomide, a golden standard in chemotherapy of brain tumors. Therefore, mapping the pathways responsible for tumorigenesis at the transcriptional level may help to determine the causes and aggressive behavior among different glial tumors. PATIENTS AND METHODS: Biopsies from patients with astrocytoma (N = 6), glioblastoma (N = 22), and meningioma (N = 14) were included in the sample set. A control group consisted of RNA isolated from healthy human brain (N = 3). The reverse-transcribed cDNAs were analyzed using the Human Cancer PathwayFinder™ real-time PCR Array in a 96-well format. The expression of 84 genes belonging to 9 signaling pathways (angiogenesis, apoptosis, cell cycle and senescence, DNA damage and repair, epithelial-to-mesenchymal transition, hypoxia, overall metabolism, and telomere dynamics) was determined for each sample. RESULTS: By determining the relative expression of selected genes, we characterized the transcriptomic profile of individual brain malignancies in the context of signaling pathways involved in tumorigenesis. We observed deregulation in 50, 52.4 and 53.6% % of the genes in glioblastomas, meningiomas and astrocytomas, respectively. The most pronounced changes with statistical significance compared to control were observed in the genes associated with epithelial-to-mesenchymal transition (CDH2, FOXC2, GSC, SNAI2, and SOX10), cellular senescence (BMI1, ETS2, MAP2K1, and SOD1), DNA repair (DDB2, ERCC3, GADD45G, and LIG4), and dynamic of telomeres (TEP1, TERF2IP, TNKS, and TNKS2). CONCLUSION: Based on the obtained data, we can conclude that individual diagnoses differ in transcriptomic profile. An individual molecular approach is therefore necessary in order to provide comprehensive and targeted therapy on multiple metabolic pathways in the diagnosis of brain tumors.

Noninvasive study of brain tumours metabolism using phosphorus-31 magnetic resonance spectroscopy.

Phosphorus-31 magnetic resonance spectroscopy (31P MRS) is currently not accepted as a diagnostic tool in the neuro-oncological practice, although it provides useful non-invasive information about biochemical processes ongoing in the intracranial tumours. This pilot study was aimed to present the diagnostic capability of the 31P MRS in brain tumour examination, even its application on clinical 1.5T MR scanner.Seven patients with brain tumorous lesions (four glioblastomas, one ependymoma, and two lung metastasis) underwent multivoxel in vivo 31P MRS performed on clinical 1.5 T MR scanner within measurement time of 20 minutes. Comparing two selected voxels, one in the tumour and the other one in the normal-appearing brain tissue, enabled to investigate their metabolic differences. Enhanced markers of membrane phospholipids synthesis (significantly increased phosphomonoesters ratios) than markers of their degradation (significantly decreased phosphodiesters ratios) manifested a higher cell proliferation ongoing in tumours. High energetic tumorous tissue demands leading to anaerobic metabolic turnover were present as a significant decline in phosphocreatine ratios and adenosine triphosphates. Intracellular pH evaluation showed a tumorous tendency to alkalize. 31P MRS enables the non-invasive metabolic characterization of intracranial tumours and thus appears to be a clinically useful method for the determination of ongoing tumour pathomechanisms (Fig. 2, Ref. 26). Keywords: brain tumour, 31P MRS, 1.5 Tesla; energetic metabolism.

Eff ect of a single asenapine treatment on Fos expression in the brain catecholamine-synthesizing neurons: impact of a chronic mild stress preconditioning.

OBJECTIVE: Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning. METHODS: Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 µl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination. RESULTS: Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA. CONCLUSIONS: Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.

Stress alters asenapine-induced Fos expression in the Meynert's nucleus: response of adjacent hypocretin and melanin-concentrating hormone neurons in rat.

OBJECTIVE: Asenapine (ASE), an atypical antipsychotic drug used in the treatment of schizophrenia, induces Fos expression in forebrain. Effect of ASE on activity of basal nucleus of Meynert (NBM) cells, a part of the striatal-cortical circuits, was studied. We were also interested to reveal whether a chronic unpredictable variable mild stress (CMS) preconditioning might affect the ASE impact. METHODS: Rats were divided into as follows: controls-vehicle, controls-ASE, stressed-vehicle and stressed-ASE groups. CMS included restrain, social isolation, crowding, swimming and cold applied for 21 days. On the 22nd day, rats were subcutaneously injected with ASE (0.3 mg/kg) or vehicle (saline 300 µl/rat), 90 min prior euthanizing. After transcardial fixation, brains were cut into 30 µm thick coronal sections. Fos protein presence, as indicator of cell activity, was detected by ABC immunohistochemistry. Hypocretin (Hcrt) and melanin-concentrating hormone (MCH) containing cells were visualized with fluorescent dyes. RESULTS: ASE induced significant increase in Fos expression in NBM in both controls and CMS preconditioned rats in comparison with the related vehicle-treated controls. CMS preconditioning, however, significantly lowered the Fos response to ASE in NBM. From Hrct and MCH cells, only Hcrt ones displayed Fos presence in response to ASE. DISCUSSION: This study demonstrates for the first time that ASE may target a special group of cells occupying NBM, which effect can be modulated by CMS preconditioning. This finding extends a view that ASE impact may extend beyond the classical forebrain target areas common for the action of all antipsychotics and might be helpful in the identification of sites and side effects of its therapeutic actions.

Effect of Asenapine on the Activity of Hypocretin Neurons in Normal and Unpredictable Mild Stress Preconditioned Rats.

Asenapine (ASE) is a novel atypical antipsychotic used in schizophrenia treatment. Here, the effect of ASE on Fos expression in hypocretin (Hcrt) neurons in medial and lateral portions of the lateral hypothalamus (LH) and the effect of chronic unpredictable variable mild stress (CMS) preconditioning were studied. CMS consisted of restraint, social isolation, crowding, swimming, and cold and lasted 21 days. The rats were sacrificed on day 22, 90 min after a single injection of vehicle (saline 300 µl/rat subcutaneously--s.c.) or ASE (0.3 mg/kg s.c.). Control (CON), ASE, CMS, and CMS+ASE groups were used. Fos protein was visualized by the avidin biotin peroxidase technique, while Hcrt perikarya by fluorescent dye. Fos/Hcrt co-localizations were evaluated under parallel light and fluorescent illuminations. In the single Fos expression assessment, the Fos number was significantly higher in the medial in comparison with the lateral LH portion in each group. No differences in Fos amount were observed between the individual groups within the medial and lateral LH portions. In the Fos/Hcrt co-localization assessments, ASE significantly reduced the number of Fos/Hcrt neurons in the medial, but not lateral, LH portion in ASE and CMS+ASE groups. CMS only slightly contributed to the inhibitory effect of ASE in the CMS+ASE groups. The present data show as the first that ASE may reduce the activity of Hcrt cells in the medial LH portion, which might correspond with the relatively low weight gain liability of ASE. CMS preconditioning did not significantly interfere with this impact of ASE.

Impact of repeated asenapine treatment on FosB/ΔFosB expression in neurons of the rat central nucleus of the amygdala: colocalization with corticoliberine (CRH) and effect of an unpredictable mild stress preconditioning.

OBJECTIVES: FosB/ΔFosB expression in the central amygdalar nucleus (CeA) in response to repeated asenapine (ASE) treatment (an atypical antipsychotic used for the treatment of schizophrenia) was studied in normal rats and rats preconditioned with chronic unpredictable variable mild stress (CMS). The goal of this study was to reveal whether repeated ASE treatment for 14 days may: 1) induce FosB/ΔFosB expression in the amygdala, 2) activate CRH-synthesizing neurons in the CeA, and 3) interfere with 21 days lasting concomitant CMS preconditioning. METHODS: Four groups of animals were studied: controls and ASE-, CMS-, and CMS+ASE-treated ones. CMS consisted of the restrain, social isolation, crowding, swimming, and cold and lasted 21 days. The ASE and CMS+ASE groups were from the 7th day of the experiment treated with ASE (0.3 mg/kg, subcutaneously - s.c.) twice a day, i.e. together for 14 days. Controls and CMS groups were treated with saline (300 µl/rat, s.c.) twice a day for 14 days. All the animals were sacrificed on the 22nd day, i.e. 16-18 hours after the last treatments. Single FosB/ΔFosB, FosB/ΔFosB colocalizations with CRH, and CRH immunolabeled perikarya were investigated in the CeA using a combined light and fluorescent immunohistochemistry. RESULTS: The distribution aspect of the black FosB/ΔFosB profiles was homogeneous over the whole CeA and no significant differences in the number of FosB/ΔFosB profiles between the individual groups of the rats really occurred. The level of colocalization pattern of FosB/ΔFosB in CRH perikarya was also very similar between the individual groups and in each case it reached approximately 10% of double-labeling. No differences were also seen in the number of CRH immunolabeled perikarya. The density of CRH nerve projections within the CeA was very alike in the individual groups of animals investigated. CONCLUSIONS: The study provides a new anatomical/functional finding about the lack of the stimulatory effect of the repeated ASE treatment on the expression of FosB/ΔFosB, FosB/ΔFosB/CRH colocalizations, and CRH immunolabeled perikarya number in the CeA. In addition, CMS preconditioning itself neither stimulated nor inhibited FosB/ΔFosB expression, nor altered the impact of ASE on the activity of CRH neurons in the CeA.

Effect of ghrelin receptor agonist and antagonist on the activity of arcuate nucleus tyrosine hydroxylase containing neurons in C57BL/6 male mice exposed to normal or high fat diet.

Catecholamines participate in the food intake regulation, however, there are no literature data available, dealing with the activity of tyrosine hydroxylase (TH) neurons in response to stimulation or inhibition of GHS-R (growth hormone secretagogue receptor) in the hypothalamic arcuate nucleus (ARC). The present study was focused to reveal whether [Dpr(N-octanoyl) 3ghrelin], a stable GHS-R agonist, itself in doses of 5 or 10 mg/kg (s.c.) or in combination with GHS-R receptor antagonist ([DLys3]GHRP-6) in dose of 10 mg/kg (s.c.), may affect the activity of ARC TH-containing neurons in C57BL/6 male mice fed either with standard (SD) or high fat diet (HFD) that developed a diet-induced obesity (DIO). The data of the present study clearly indicate that both doses of GHS-R agonist stimulated food intake in SD mice and GHS-R antagonist significantly reduced GHS-R agonist orexinergic effect in SD mice and suppressed the voluntary food intake in HFD mice. Both doses of the GHS-R agonist stimulated Fos expression in ARC neurons in both diet groups of mice which was not abolished by GHS-R antagonist pretreatment. Moreover, both doses of the GHS-R agonist significantly influenced the activation of TH neurons in the ARC of SD mice. The GHS-R antagonist also significantly increased TH neurons activation after GHS-R agonist although this effect was less powerful in HFD mice. This is the first study demonstrating response of local ARC TH neurons to peripherally applied GHS-R agonist and antagonist. The present data point out that the response of TH neurons to GHS-R agonist and antagonist is different in normal and DIO mice and extend our knowledge about the further ARC neuronal phenotype responding to peripheral ghrelin. To bring insight into the understanding of the functional significance of the activated TH neurons in ARC, in the context of the ghrelin peripheral increase, further studies are required.

A new approach of light microscopic immunohistochemical triple-staining: combination of Fos labeling with diaminobenzidine-nickel and neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes.

OBJECTIVE: The aim of the present study was to introduce a new approach of the light microscopic immunohistochemical triple-staining enabling to study the differences in the activity of at least two different phenotypes of neurons on the same histological section. For this purpose combination of Fos (a product of the immediate early gene) labeling with nickel intensified diaminobenzidine (DAB-Ni) and two neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes on cryo-processed 35-40 µm thick free-floating brain sections was selected. METHODS: The parallel occurrence of three antibodies studied, i.e. Fos, hypocretin (HCRT), and melanin-concentrating hormone (MCH), was studied by a new methodic approach utilizing combination of Fos immunolabeled with DAB-Ni and HCRT and MCH labeled with Alexa488 and Alexa555 fluorescent dyes, respectively. Fos stimulation was induced by a single immobilization (IM0) for 120 min. Then, the rats were sacrificed, the brains removed, soaked with 30% sucrose in 0.1 M phosphate buffer (PB), cryo-sectioned throughout the hypothalamus into 35-40 µm thick coronal sections, collected, and washed in the same buffer for 10-15 min. Fos was revealed by avidin-biotin-peroxidase (ABC) complex and visualized by diaminobenzidine chromogen containing nickel chloride salt. HCRT and MCH neurons were visualized by the above mentioned fluorescent dyes. Evaluation of the Fos and fluorescent staining was performed in the computerized Axo Imager Carl Zeiss microscope using light and fluorescent illuminations. RESULTS: All the antibodies used showed clear immunoreactive staining. Fos staining occurred in the form of black color located in the cell nuclei. HCRH and MCH neuropeptides showed clear green and red fluorescence in the cell perikarya, respectively. The final merged picture showed Fos protein in the activated green HCRT or red MCH neurons in the form of white nuclei. CONCLUSIONS: The present study clearly demonstrate that the combination of Fos labeling with DAB-Ni and neuropeptides labeled with Alexa488 and Alexa555 on cryo-processed 35-40 µm thick free-floating brain sections is an excellent approach providing further advantages for quick and reproducible triple immuno-staining enabling to compare the activity of at least two phenotypes of neurons on the same section. KEYWORDS: Alexa488 and Alexa555 fluorescent dyes, Fos, hypocretin, melanin-concentrating hormone, cryostat sections, triple labeling immunohistochemistry, rat.