Cultured C6 Glioma Cells Research Articles

ID: 204674 Electrical Stimulation of Glioma Cells: Effect on Neurotransmitters and Intracellular Nitric Oxide, Chloride, and Calcium

In vitro glial cell models can be used to assess changes that might correlate with putative molecular effects that electrical stimulation (ES) could have on neuroglia. Cell models allow for faster and cheaper experiments than animal models due to the potential to easily vary the conditions, reduce the number of variables, and closely monitor specific effects. We previously showed that modulation of gene expression in cultured C6 glioma cells, an astrocyte model system, was dependent on the anodic content of electrical stimulation (ES)1. Expanding on this research, we assessed molecular changes in response to passively balanced cathodic ES pulses in the presence of neurotransmitters.

STEM-19. LIVE DETECTION OF NEURAL STEM AND GLIOBLASTOMA CELLS BY A LUMINESCENT CONJUGATED OLIGOTHIOPHENE DERIVATIVE

Abstract Glioblastoma (GBM) is an aggressive nervous system tumor with a mean survival time of 12-14 months. Cells with neural stem cell-like properties can be derived from GBM tumors. These cells seem to escape conventional irradiation treatment, chemotherapy, and surgery, and may play a crucial role for relapse. It is therefore urgent to develop novel approaches for reliable detection of neural stem cell-like cells in GBM. Here we report a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), for non-invasive and non-amplified real-time detection of live human patient-derived GBM cells and embryonic neural stem/progenitor cells (NSPCs). Within a maximum of 10 minutes after administration of the molecule in vitro, in the existing media, fluorescence emission was observed without any modulation of the cells or additional vehicle, resulting in efficient detection of cytoplasmic luminescent signal in NSPCs or GBM cells from rodents and humans, detectable at Alexa488/GFP wavelength. GlioStem is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. In vitro, GlioStem was shown to identify fetal cortical NSPCs from rat (FGF2-expanded), embryonic stem cell-derived NSPCs from mouse (FGF2/EGF-expanded), and FGF2-exposed C6 glioma cell cultures from rat, but not any other cell types investigated. Cell sorting experiments of patient-derived, FGF2/EGF-expanded GBM cells demonstrated that GlioStem in addition to NSPC-markers like Nestin and Sox2 labeled the same population (overlap > 90%) of cells as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO GlioStem is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.

Effects of low-dose, short-duration periods of asymmetric radiation on colony formation of C6 glioma cell cultures

Background/Aim: Previous studies on fractionation in radiation therapy have been mainly based on applying equal doses over at least 6 h. The main purpose of fractionation is to increase normal tissue tolerance rather than tumor sensitivity. Thus, one can apply higher doses to the tumor. In contrast, new molecular studies indicate that high and low doses of radiation act by different mechanisms. This study was conducted to investigate the radiobiological effect of asymmetrical radiation doses.
 Methods: This is an experimental study done in vitro with a G6 glioma cell line to investigate the responses when C6 glioma cells are irradiated with single doses of 30 and 230 cGy using an orthovoltage therapy device or doses split into 30 and 200 and 115 and 115 cGy within periods of 15 and 30 min. A total of 5 × 103 cells were transferred to polyethylene culture flasks for colony formation. A cluster containing more than 30 cells was considered a new colony.
 Results: A single dose of 230 cGy caused a 56.8% reduction in colony formation. However, when 230 cGy was divided over 15- and 30-min periods in fractions of 30 and 200 cGy, colony formation was significantly reduced compared to the control group (68.13% and 52.64%, P = 0.030, respectively). This effect continued when the radiation dose was divided into equal fractions (115 and 115 cGy) with periods of 15 and 30 min (42.60%, P = 0.021 and 20.77%, P = 0.008, respectively).
 Conclusion: According to these results, (i) short interval (15 and 30 min) fractionation significantly reduces colony formation compared to a single equal dose; and (ii) the protective mechanisms activated in cell response probably vary at different radiation doses and different fractions.

The Effects of Methanol Extracts of Hyssopus officinalis on Model of Induced Glioblastoma Multiforme (GBM) in Rats.

Given the complexity of pathophysiological processes of brain tumors, ineffective therapies, and high mortality rate, new therapeutic options with less toxicity are necessary. Hyssopus officinalis (hyssop) is an aromatic plant with important biological activities. The aim of this study is to assess the anti-cancer effect of hyssop extract on damages of glioblastoma multiforme. In this study, total flavonoids, phenolic content, and quantification of phenolic compound of hyssop extracts were analyzed. In vitro antioxidant properties of hyssop extract were also examined. In addition, cell viability, apoptosis, and cell cycle were evaluated in C6 glioma cell culture. In vivo, the rats were divided randomly into four main groups: intact, control, vehicle, and treatment groups. 1 × 106 C6 rat glioma cells were implanted into the right caudate nucleus of the rat's brain. The treatment group received the methanol extract of hyssop (100mg/kg) for 7days. Evolution of locomotor activity, tumor volume, survival rate, activities of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), vascular endothelial growth factor (VEGF) expression, TUNEL-positive cells, p53 and p21 mRNA expression, and histological alterations were performed. The results showed that the methanol extract of hyssop increased the apoptosis and reduced the cell division of C6 glioma cells in cell culture. Moreover, methanol extract decreased the tumor volume and prolonged survival. Also, the activity of SOD and CAT enzymes was reduced in tumor tissue and enhanced in surrounding tissue. TUNEL-positive cells were increased in methanol extract of hyssop group. The expression of p53 and p21 mRNA was upregulated in the treatment group. Moreover, the histological analysis indicated a considerable decrease in invasion of tumor cells and inflammation in the hyssop-treated rats. According to the achieved results, it can be stated that hyssop has sufficient potential to inhibit damage of brain tumors, at least in part, by affecting the oxidative stress and cell proliferation pathways.

Valeriana officinalis Counteracts Rotenone Effects on Spreading Depression in the Rat Brain in vivo and Protects Against Rotenone Cytotoxicity Toward Rat Glioma C6 Cells in vitro.

Astrocytes can protect neurons against oxidative stress and excitability-dependent disorders, such as epilepsy. Valeriana officinalis has been used as anticonvulsant and can exert an antioxidant effect, which may underlie its opposing action against the toxic effects of the pesticide rotenone. We investigated the V. officinalis/rotenone interaction in the cortical spreading depression (CSD), a phenomenon that depends upon brain excitability (in vivo model). In addition, we analyzed the protective action of V. officinalis against the cytotoxic effects of rotenone in cultures of rat C6 glioma cells (in vitro model). For the CSD study, Wistar rats received either V. officinalis (250 mg/kg/day via gavage for 15 days; n = 8) or 10 mg/kg/day rotenone via subcutaneous injections for 7 days (n = 7), or they received both substances (n = 5). Two control groups received either saline (vehicle for V. officinalis; n = 8) or 1% Tween-80 aqueous solution (vehicle for rotenone; n = 9). After treatment, CSD was recorded for 4 h. The rotenone- and V. officinalis-treated groups presented, respectively, with lower (2.96 ± 0.14 mm/min), and higher CSD propagation velocity (3.81 ± 0.10 mm/min) when compared with the controls (Tween-80, 3.37 ± 0.06 mm/min and saline, 3.35 ± 0.08 mm/min; p < 0.05). The rotenone plus V. officinalis-treated group displayed a CSD velocity (3.38 ± 0.07 mm/min) that was similar to controls. In line with these results, in vitro experiments on rat glioma C6 cells revealed a protective effect (MTT assay) of V. officinalis against rotenone-induced cytotoxicity. These results suggest the therapeutic potential of V. officinalis for treating neurological diseases involving redox imbalance and astrocyte dysfunction.

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

Malignant Glioma Therapy by Vaccination with Irradiated C6 Cell-Derived Microvesicles Promotes an Antitumoral Immune Response.

Glioblastoma is the most common and malignant tumor of the CNS, with a mean survival of 14 months after diagnosis. Its unfavorable prognosis reveals the need for novel therapies. It is known that radiation can induce a systemic antitumor effect. Tumor cells produce and release microvesicles in response to cell damage such as radiation. Microvesicles contain a plethora of bioactive molecules, including antigens involved in modulation of the immune response. In this study, we characterized and evaluated irradiated C6 cell-derived microvesicles as a therapeutic vaccination in C6 malignant glioma. Cultured C6 glioma cells were irradiated with a single dose of 50 Gy to obtain the microvesicles. Subcutaneous implantation of C6 cells was performed when the tumor reached 2 cm in diameter, and non-irradiated and irradiated C6 cell-derived microvesicles were administered subcutaneously. Tumor growth, apoptosis, and immunophenotypes were determined. Reduction of tumor volume (more than 50%) was observed in the group treated with irradiated C6 cell-derived microvesicles compared with the control (p = 0.03). The percentages of infiltrative helper, cytotoxic, and regulatory T lymphocytes as well as apoptotic cells were increased in tumors from immunized rats compared with controls. These findings make microvesicle-based vaccination a promising immunotherapeutic approach against glioblastoma.

Erucic acid, a component of Lorenzo's oil and PPAR-δ ligand modifies C6 glioma growth and toxicity of doxorubicin. Experimental data and a comprehensive literature analysis

BackgroundPPAR-δ is a transcription factor which has crucial roles in stimulating oligodendroglial differentiation and myelination and its activation was also shown to differentiate malignant C6 glioma cells into oligodendrocytes. ObjectiveOne of the ligands of PPAR-δ is erucic acid (EA), an edible omega-9 fatty acid consumed more by Asian populations and exists highly in Chinese womens milk. There exist epidemiological evidence that pediatric brain tumor incidence is among the lowest in the Chinese population. EA is also an ingredient of Lorenzo's oil used against adrenoleukodystrophy, a pediatric demyelinating disease. EA was inappropriately assumed as a strong cardiotoxin based on Spanish oil syndrome, caused by toxic-aniline dye refined rapeseed oil. In this study, we studied whether EA is capable to block growth of C6 glioma cells and modify cardiotoxicity of doxorubicin. Materials and methodsWe studied effects of EA on the 3-dimensional appearance of the adherent cells, soft agar colony formation and S-phase in the 3-dimensional spheroids in C6 glioma cell cultures. We also investigated the effects of EA on hepatic and cardiac toxicity of doxorubicin. ResultsEA decreased in vitro growth of C6 glioma cells at therapeutically achievable concentrations. EA effects were more prominent in 3D-assays (soft agar colonies and spheroids) and induced cell fusions in monolayer cultures. EA decreased S-phase inhibitory potency of doxorubicin (DOX), yet augmented its efficacy to induce a senescent morphology (as assessed by scanning electron microscopy) in monolayer and to increase iNOS and eNOS expression in spheroids. In our study, EA reduced DOX-induced necrosis in mice heart and liver and induced healthier morphology of heart mitochondria (as assessed by transmission electron microscopy); yet intercalated disks (ID) were more disturbed with DOX + EA. ConclusionsBoth the antitumor and cardiac effects of EA may associate with the cell-to-cell contact mechanisms. Combining systemic EA with intrathecal DOX-chemotherapy via Ommaya reservoirs may reduce DOX concentrations in systemic circulation, hinder toxic interactions with EA and induce selective kill of glioma cells.

The Protective Effect of Lonicerae flos Extract on Cultured C6 Glioma Cells Damaged by Aluminum of Dementia Inducer

치매유발제인 염화알루미늄(aluminum chloride, $AlCl_3$ )의 세포독성을 대뇌교종세포인 C6 glioma 세포를 대상으로 산화적 손상측면에서 조사하였으며, 또한 $AlCl_3$ 의 세포독성에 대한 금은화(Lonicerae flos, LF) 추출물의 영향을 세포생존율을 비롯한 DPPH-라디칼 소거능, superoxide anion-radical (SAR) 제거...

Temperature-amplitude coupling for stable biological rhythms at different temperatures.

Most biological processes accelerate with temperature, for example cell division. In contrast, the circadian rhythm period is robust to temperature fluctuation, termed temperature compensation. Temperature compensation is peculiar because a system-level property (i.e., the circadian period) is stable under varying temperature while individual components of the system (i.e., biochemical reactions) are usually temperature-sensitive. To understand the mechanism for period stability, we measured the time series of circadian clock transcripts in cultured C6 glioma cells. The amplitudes of Cry1 and Dbp circadian expression increased significantly with temperature. In contrast, other clock transcripts demonstrated no significant change in amplitude. To understand these experimental results, we analyzed mathematical models with different network topologies. It was found that the geometric mean amplitude of gene expression must increase to maintain a stable period with increasing temperatures and reaction speeds for all models studied. To investigate the generality of this temperature–amplitude coupling mechanism for period stability, we revisited data on the yeast metabolic cycle (YMC) period, which is also stable under temperature variation. We confirmed that the YMC amplitude increased at higher temperatures, suggesting temperature-amplitude coupling as a common mechanism shared by circadian and 4 h-metabolic rhythms.

Bimodal modulation of store-operated Ca2+ channels by clozapine in astrocytes

Clozapine (Clz) and olanzapine (Olz) are second generation (atypical) antipsychotics, used widely for treating schizophrenia and bipolar disorder. These drugs share multiple sites of actions, however their mechanisms remain incompletely understood. Here, we analyzed the effects of these drugs on primary cultures of rat cortical astrocytes and C6 glioma cells using fura-2-based Ca2+ imaging. C6 cells, but not cortical astrocytes, express the serotonin 2A receptor subtype, which couples to phospholipase C. Clz (1μM) significantly blocked serotonin-induced Ca2+ transients in C6 cells, consistent with known antagonistic actions of Clz. Interestingly, at higher concentrations (>10μM), Clz but not Olz increased intracellular Ca2+ concentrations in both cortical astrocytes and C6 cells. This Clz-induced Ca2+ increase was concentration-dependent and completely blocked by removal of extracellular Ca2+ using ethylene glycol tetraacetic acid (EGTA). Furthermore, 2-aminoethyl diphenylborinate or SKF-96365, blockers for store-operated Ca2+ channels, significantly inhibited the Clz-induced Ca2+ increase. Therefore, we analyzed the effects of Clz and Olz during Ca2+ re-entry through store-operated Ca2+ channels, which was maximized following depletion of internal Ca2+ stores by thapsigargin and EGTA. The results demonstrated that Clz decreased Ca2+ re-entry through store-operated Ca2+ channels in cortical astrocytes and C6 cells whereas Olz failed to modulate the Ca2+ re-entry. These results suggest Clz-specific bimodal actions via store-operated Ca2+ channels in astrocytic cells. Since intracellular Ca2+ homeostasis in astrocytes is an important determinant for neighboring synaptic signal transmission, our results may explain Clz-specific adverse effects or differential actions between Clz and Olz reported in the treatment of psychiatric disorders.

P08.03 Erucic acid, a component of Lorenzo’s oil used in treatment of adrenoleukodystrophy, acts antineoplastic in C6 glioma cell culture

P08.03 Erucic acid, a component of Lorenzo’s oil used in treatment of adrenoleukodystrophy, acts antineoplastic in C6 glioma cell culture

Induction of proline-rich tyrosine kinase 2 activation-mediated C6 glioma cell invasion after anti-vascular endothelial growth factor therapy

BackgroundAnti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF).MethodsThe effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting.ResultsBevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats.ConclusionsThese results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.

Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION&amp;ndash;EGF) for targeting brain tumors

Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION–EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION–EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION–EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION–EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION–EGF conjugates in animals provided receptor-mediated targeted delivery across the blood–brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION–EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.

Adaptive Changes in the Heterologous Desensitization Event after Histamine Activates its H1 Receptor in C6 Glioma Cells

The possible role of protein kinase C (PKC) linked to the heterologous desensitization of histamine H1 and other receptors was investigated. Receptor activators, such as ATP, PAF, and bradykinin, or calcium ionophore A-23187 were employed for study in the cultured C6 glioma cells. Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free calcium concentration ([Ca^(2+)](subscript i)) were monitored in response to histamine H1 or other receptor activations. Histamine stimulated the release of Ca^(2+) from intracellular Ca^(2+) stores and Ca^(2+) influx across the plasma membrane. Under pre-activation of PKC with the phorbol ester β-phorbol-12, 13 dibutyrate (PMA, 10 nM), it caused an attenuation of histamine (100 μM), ATP (100 μM), PAF (1 μM), or bradykinin (1 μM)-induced [Ca^(2+)](subscript i) increase. The relative potency order of attenuation was histamine (92.2%) ＞ ATP (83.3%) ＞ PAF (50.2%) ＞ bradykinin (48.0%) ＞ A23187 (0.2%) in the calcium movement. Similar trend was observed in the histamine pretreatment case among these agents. In this study, 100 nM A23187 (its action is independent of protein kinase C activity), was not found to have a heterologous desensitization in histamine- or PMA-induced calcium movement. Hence PKC activator PMA, and its inhibitor staurosporine could cause significant effects on this heterologous desensitization event. It is proposed that PKC phosphorylating the substrate proteins plays an important role in this shortterm desensitization of histamine acting on histamine H1 receptors in the C6 glioma cells. Our data support the claim that PKC phosphorylates its substrate of the inositol 1, 4, 5-trisphosphate receptor (IP3R) and decreases the mobilization of [Ca^(2+)](subscript i) in these cells, which linked to homologous or/and heterologous desensitization.

Induction of β-Glucuronidase Release by Cytostatic Agents in Small Tumors

Extracellular β-glucuronidase (β-GUS) in tumors has been investigated as a target enzyme for prodrug therapy. However, despite encouraging preclinical results, animal studies also indicate that the success of prodrug therapy might be limited by the insufficient prodrug-converting enzyme activity, especially in small tumors. We hypothesized that a single dose of a cytostatic drug might induce the release of β-GUS in small tumors, resulting in increased levels of extracellular β-GUS and consequently a higher efficacy of the prodrug treatment. Here we examine the extent of β-GUS release in small C6 glioma tumors after a single treatment of doxorubicin (DOX), carmustine (BCNU) and tumor necrosis factor α (TNF-α) with positron emission tomography (PET) and the tracer 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate, [(18)F]FEAnGA, which has been proven to be selective for extracellular β-GUS. Induction of β-GUS release was first investigated in cultured C6 glioma cells. In addition, a [(18)F]FEAnGA PET study was performed in C6 tumor-bearing rats 48 h after a single treatment with different cytostatics to evaluate the extent of β-glucuronidase release. The cleavage of [(18)F]FEAnGA by β-GUS was analyzed in tumor homogenates. The induction of tumor necrosis and leukocyte infiltration was confirmed by histochemical analysis and flow cytometry. The in vitro studies indicated that all treatments resulted in a decline of viable cells and an increase of extracellular β-GUS activity. PET studies confirmed that β-GUS was released in vivo and the distribution volume of the PET tracer [(18)F]FEAnGA in C6 gliomas was increased significantly by 15-70%, depending on the treatment. Histochemical analysis of the tumors indicated that carmustine and TNF-α treatment caused a larger necrotic area with the absence of infiltrating immune cells, whereas doxorubicin induced an increase in leukocyte infiltration. These results were confirmed by flow cytometry. In conclusion, the present study demonstrates that a single dose of a cytostatic agent is able to increase the release of β-GUS. The release in β-GUS can be monitored by [(18)F]FEAnGA PET in a noninvasive manner. This study may open the way to a two-step chemotherapy-prodrug approach, in which tumors are treated with a single dose of a cytostatic drug prior to prodrug treatment.

The Circadian Gene Expression of Per1 and Per2 and Their Influence on Radiotherapeutic Sensitivity of Glioma In Vitro

Per2 plays a key role in regulating the circadian rhythm in mammals. However, the circadian clock gene expression of Per1 and Per2 and its influence on radiotherapeutic sensitivity of C6 glioma cells in vitro have not been explored. Aim: To investigate the rhythm expression of circadian gene Per1 and Per2, and examine the influence on radiotherapeutic sensitivity of two important clock genes in C6 glioma cells. Materials &amp; methods: The cultured C6 glioma cells and NIH3T3 cells were stimulated by phorbol 12-myristate 13-acetate (PMA). The expression of Per1 and Per2 at the indicated times were examined with a method for the absolute quantification of cDNA using real-time PCR. The cultured cell were given x-irradiation at the indicated times and the cell-cycle, apoptosis and proliferation were examined by flow cytometry. Results: We report here that PMA treatment of C6 rat glioma cells induces circadian expression of Per2, and that during periods of high expression, cells are blocked at the G2/M transition and are more sensitive to x-irradiation. PMA treatment of NIH3T3 cells induced circadian expression of Per1 and Per2, but high Per expression did not block the cell cycle or render the cells more sensitive to irradiation. Conclusion: Our results suggest that Per2 expression may increase the efficacy of radiotherapy against glioma.

Generation of Monoclonal Antibodies to Recombinant Vascular Endothelial Growth Factor

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.

ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spec- troscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O2 •- /HO2 • ,H O • , and H2O2 generated upon 4-MeV X-ray irradiation of 6.4 lM silicon nanoparticle aqueous suspensions were on the order of 10 lM per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic 1 O2 was generated only in irradi- ation experiments containing the particles. The particle surface became oxidized to SiO2 and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experi- mental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradia- tion-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of\5 nm size have the potential to be used as radiosensitizers for improv- ing the outcomes of cancer radiotherapy. Their capa- bility of producing 1 O2 upon X-ray irradiation opens