Aloe vera and Fermented Extracts Exhibit an Anti-Inflammatory Effect on Human Glioblastoma/Astrocytoma U373 MG Cells.

The anti-inflammatory effects of Aloe vera (AV), polysaccharide extract from AV, and extracts from the digestion and colonic fermentation of AV were evaluated using an immortal astrocyte cell line (U373 MG) that develops a neuro-inflammatory profile. Cell viability and inflammatory markers were assessed after stimulation with neuropeptide substance P (SP) that activates the pro-inflammatory MAPK (mitogen-activated protein kinase) pathway. Cell viability after SP treatment was over 50% at 10 mg/mL AV, polysaccharide extract from AV, extracts from the digestion: non-digestible fraction of AV non-digestible fraction of polysaccharide extract from AV and extracts from the colonic fermentation of AV, at 4 and 24 h. Moreover, cells exposed to SP and treated with these extracts showed lower protein-activated ERK1/ERK2 (extracellular signal-regulated kinases 1 and 2), p38 (MAPK protein p38), and NFκB (nuclear factor κB) levels with respect to the SP-stimulated control. Inflammation inhibition by extracts of polysaccharide extract from AV and extracts from the colonic fermentation of AV, at 24 h in the study of p38 was not as statistically significant in ERK1/ERK2 and NFκB. Nevertheless, there was a significant decrease (p < 0.05) in pro-inflammatory cytokine IL-6 levels in cells exposed to all samples. Samples with extracts from the colonic fermentation of AV, at 4 or 24 h showed the highest inhibitory effect on IL-6 production.

4-Methoxydalbergione is a potent inhibitor of human astroglioma U87 cells in vitro and in vivo.

Astroglioma is the most common primary tumor in the central nervous system without effective treatment strategies. Temozolomide (TMZ) is a chemotherapeutic drug to treat astroglioma but exhibits low potency and has side effects. Therefore, there is an urgent need to develop new compounds to treat astroglioma. Dalbergia sissoo Roxb was the source of Dalbergia odorifera in traditional Chinese medicine (TCM) and has been clinically used as an anti-tumor medicine. 4-Methoxydalbergione (4MOD) is purified from Dalbergia sissoo Roxb., and shows an inhibitory effect on osteosarcoma, but its effects on astroglioma have not been reported. Here, we evaluate its anti-astroglioma effects on both in vitro and in vivo models. In cultured astroglioma U87 cells, 4MOD inhibited cell proliferation and induced cell apoptosis in a time- and concentration-dependent manner. Compared with TMZ, 4MOD exhibited a tenfold greater potency of anti-astroglioma effects. 4MOD effectively stalled the cell cycle in G2 phase. Transcriptome sequencing (RNA-seq) showed that 4MOD upregulated 158 genes and downregulated 204 genes that are mainly enriched in cell membrane, cell division, cell cycle, p53, TNF, and MAPK signaling pathways, which may underlie its anti-tumor mechanisms. In a nude mouse xenograft model transplanted with U87 cells, 10 mg/kg 4MOD slowed down tumor growth rate, while at 30 mg/kg dose, it reduced tumor size. Collectively, this study demonstrates that 4MOD is a potent native compound that remarkably inhibits U87 astroglioma growth in both in vitro and in vivo models.

Yokukansan, a Japanese Herbal Medicine, Suppresses Substance PInduced Production of Interleukin-6 and Interleukin-8 by Human U373 MG Glioblastoma Astrocytoma Cells.

BACKGROUND: Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear. OBJECTIVE: This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP). METHODS: We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p < 0.05. RESULTS: Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells. CONCLUSION: Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Sigma-2 receptor/TMEM97 agonist PB221 as an alternative drug for brain tumor.

BACKGROUND: There are limited effective drugs that can reach the brain to target brain tumors, in particular glioblastoma, which is one of the most difficult cancers to be cured from. Because the overexpression of the sigma-2 receptor is frequently reported in glioma clinical samples and associated with poor prognosis and malignancy, we herein studied the anti-tumor effect of the sigma-2 receptor agonist PB221 (4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperidine) on an anaplastic astrocytoma tumor model based on previous encouraging results in pancreatic cancer and neuroblastoma SK-N-SH cells. METHODS: The expression of the sigma-2 receptor, transmembrane protein 97 (TMEM97), in ALTS1C1 and UN-KC6141 cell lines was measured by RT-PCR and quantitative RT-PCR. The binding of sigma-2 receptor fluorescent ligands PB385 (6-[5-[3-(4-cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronaphthalen-5-yloxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine) and NO1 (2-{6-[2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one-5-yloxy]hexyl}-5-(dimethylamino)isoindoline-1,3-dione) was examined by flow cytometry and the fluorescent plate reader. The antitumor activity of PB221 was initially examined in the murine brain tumor cell line ALTS1C1 and then in the murine pancreatic cell line UN-KC6141. The potential therapeutic efficacy of PB221 for murine brain tumors was examined by in vitro migration and invasion assays and in vivo ectopic and orthotopic ALTS1C1 tumor models. RESULTS: The IC50 of PB221 for ALTS1C1 and UN-KC6141 cell lines was 10.61 ± 0.96 and 13.13 ± 1.15 µM, respectively. A low dose of PB221 (1 µM) significantly repressed the migration and invasion of ALTS1C1 cells, and a high dose of PB221 (20 µM) resulted in the apoptotic cell death of ALTS1C1 cells. These effects were reduced by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine, suggesting mitochondrial oxidative stress is involved. The in vivo study revealed that PB221 effectively retarded tumor growth to 36% of the control tumor volume in the ectopic intramuscular tumor model and increased the overall survival time by 20% (from 26 to 31 days) in the orthotopic intracerebral tumor model. CONCLUSIONS: This study demonstrates that the sigma-2 receptor agonist PB221 has the potential to be an alternative chemotherapeutic drug for brain tumors with comparable side effects as the current standard-of-care drug, temozolomide.

Surgical and molecular considerations in the treatment of pediatric thalamopeduncular tumors.

OBJECTIVE Thalamopeduncular tumors are a group of pediatric low-grade gliomas that arise at the interface of the thalamus and brainstem peduncle. They typically occur within the first 2 decades of life, presenting with progressive spastic hemiparesis. Treatment strategies, including surgical intervention, have varied significantly. The authors present their experience in the treatment of 13 children, ages 2-15 years, with non-neurofibromatosis-related pilocytic astrocytomas located in the thalamopeduncular region. METHODS Between 2003 and 2016, 13 children presenting with progressive spastic hemiparesis due to a pilocytic astrocytoma at the interface of the thalamus and cerebral peduncles were identified. Medical records were reviewed retrospectively for clinical, radiological, pathological, and surgical data. Formalin-fixed, paraffin-embedded tissue was obtained for 12 cases and tested for KIAA1549-BRAF fusion and BRAF V600E point mutation. RESULTS On preoperative diffusion tensor imaging tractography (performed in 12 patients), the ipsilateral corticospinal tract was displaced laterally in 1 case (8.3%), medially in 1 case (8.3%), anterolaterally in 10 cases (83%), and posteriorly in no cases. Ten patients underwent resection via a transtemporal, transchoroidal approach, which was chosen to avoid further damage to motor function in cases of tumors that caused anterolateral or medial corticospinal tract displacement. With this approach, complications included hemianopia, oculomotor palsy, and tremor at a rate of 50%. Among the 12 patients with obtainable follow-up (mean 50.9 months), none received adjuvant therapy, and only 2 (17%) experienced recurrence or progression. KIAA1549-BRAF fusions were present in 10 cases (83%), while BRAF V600E was absent (0%). The 2 fusion-negative tumors had clinical features atypical for the series, including multi-focality and infiltration. CONCLUSIONS Transcortical, transchoroidal resection of thalamopeduncular tumors through the middle temporal gyrus allows for a high rate of gross-total resection and cure. Diffuse tensor tractography is a critical component of the preoperative planning process to determine the location of white matter tracts in proximity. Molecular status may correlate with clinical features, and the presence of BRAF lesions offers an additional target for future novel therapeutics.

Maintenance of Stemlike Glioma Cells and Microglia in an Organotypic Glioma Slice Model.

BACKGROUND: The therapeutic resistance of gliomas is, at least in part, due to stemlike glioma cells (SLGCs), which self-renew, generate the bulk of tumor cells, and sustain tumor growth. SLGCs from glioblastomas (GB) have been studied in cell cultures or mouse models, whereas little is known about SLGCs from lower grade gliomas. OBJECTIVE: To compare cell and organotypic slice cultures from GBs and lower grade gliomas and study the maintenance of SLGCs. METHODS: Cells and tissue slices from astrocytomas, oligodendrogliomas, oligoastrocytomas, and GBs were cultivated in (1) serum-free medium supplemented with the growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), (2) medium containing 10% serum plus EGF and bFGF (F+GF medium), or (3) medium containing 10% fetal calf serum (F medium). Maintenance of cells and cytoarchitecture was addressed, using several candidate SLGC markers (Nestin, Sox2, CD133, CD44, CD49f/integrin α6, and Notch) as well as CD31 (endothelial cells), ionized calcium-binding adapter molecule 1 (microglia), and vimentin. Cell vitality was determined. RESULTS: SLGCs were present in tissue slices from lower and higher grade gliomas. Preservation of the cytoarchitecture in slices was possible for >3 weeks. Maintenance of SLGCs required the presence of EGF/bFGF in cell and slice cultures, in which F+GF appeared superior to N medium. Constraints were observed regarding the preservation of the microglia but not of the endothelial cells. Maintenance of the microglia was improved by addition of the cytokine macrophage colony-stimulating factor. CONCLUSION: Medium supplemented with serum and growth factors EGF, bFGF, and macrophage colony-stimulating factor permits the preservation of SLGCs and non-SLGCs in the original glioma microenvironment.

Biological activity of HPLC-characterized ethanol extract from the aerial parts of Haplophyllum tuberculatum.

CONTEXT: The search for new sources of natural antioxidants from plant material may have beneficial therapeutic potential for those diseases associated with oxidative stress. The medicinal plant Haplophyllum tuberculatum (Forsskal) A. Juss. (Rutaceae) contains phenolic compounds as main phytochemicals; however, there are no reports on its antioxidant properties. OBJECTIVE: To evaluate antioxidant and cytoprotective potential of ethanol extract of Haplophyllum tuberculatum aerial parts. MATERIALS AND METHODS: Total phenol content was determined using Folin-Ciocalteu reagent; antiradical activity was measured using ORAC assay and the analysis of the major polyphenols was carried out using a HPLC-MS method. The antioxidant and cytoprotective effect were also investigated by the MTT assay and DCFH-DA method. The human astrocytoma U373-MG cell line was pretreated with ethanol extract (from 0.025 to 250 µg/mL) for 24 h, prior to 1 mM H2O2 exposure (30 min). RESULTS AND CONCLUSION: Total phenol content was 46.2 mg gallic acid/g sample and ORAC value was 1.283 µmol TE/mg sample. Chemical constituents were methoxyflavones, flavonols (mainly quercetin derivatives), cinnamic acids and benzoic acids. In cell system model of oxidative stress, pretreatments with ethanol extract at the concentrations of 2.5, 0.25 and 0.025 µg/mL significantly attenuated H2O2-induced loss in viability by 13.5, 17 and 20.5%, respectively. Furthermore, these ethanol extract concentrations markedly inhibited intracellular ROS production with IC50 0.026 µg/mL. These findings demonstrate the beneficial properties of ethanol extract of Haplophyllum tuberculatum aerial parts, rich in phenolic compounds, as antioxidant and radical scavenger ameliorating ROS-related processes and diseases such as several neurodegenerative disorders.

Application of 31P MR spectroscopy to the brain tumors.

OBJECTIVE: To evaluate the clinical feasibility and obtain useful parameters of (31)P magnetic resonance spectroscopy (MRS) study for making the differential diagnosis of brain tumors. MATERIALS AND METHODS: Twenty-eight patients with brain tumorous lesions (22 cases of brain tumor and 6 cases of abscess) and 11 normal volunteers were included. The patients were classified into the astrocytoma group, lymphoma group, metastasis group and the abscess group. We obtained the intracellular pH and the metabolite ratios of phosphomonoesters/phosophodiesters (PME/PDE), PME/inorganic phosphate (Pi), PDE/Pi, PME/adenosine triphosphate (ATP), PDE/ATP, PME/phosphocreatine (PCr), PDE/PCr, PCr/ATP, PCr/Pi, and ATP/Pi, and evaluated the statistical significances. RESULTS: The brain tumors had a tendency of alkalization (pH = 7.28 ± 0.27, p = 0.090), especially the pH of the lymphoma was significantly increased (pH = 7.45 ± 0.32, p = 0.013). The brain tumor group showed increased PME/PDE ratio compared with that in the normal control group (p = 0.012). The ratios of PME/PDE, PDE/Pi, PME/PCr and PDE/PCr showed statistically significant differences between each brain lesion groups (p < 0.05). The astrocytoma showed an increased PME/PDE and PME/PCr ratio. The ratios of PDE/Pi, PME/PCr, and PDE/PCr in lymphoma group were lower than those in the control group and astrocytoma group. The metastasis group showed an increased PME/PDE ratio, compared with that in the normal control group. CONCLUSION: We have obtained the clinically applicable (31)P MRS, and the pH, PME/PDE, PDE/Pi, PME/PCr, and PDE/PCr ratios are helpful for differentiating among the different types of brain tumors.

Semaphorin 5A and plexin-B3 regulate human glioma cell motility and morphology through Rac1 and the actin cytoskeleton.

Semaphorins are implicated in glioma progression, although little is known about the underlying mechanisms. We have reported plexin-B3 expression in human gliomas, which upon stimulation by Sema5A causes significant inhibition of cell migration and invasion. The concomitant inactivation of Rac1 is of mechanistic importance because forced expression of constitutively active Rac1 abolishes these inhibitory effects. Furthermore, Sema5A induces prominent cell collapse and ramification of processes reminiscent of astrocytic morphology, which temporally associate with extensive disassembly of actin stress fibers and disruption of focal adhesions, followed by accumulation of actin patches in protrusions. Mechanistically, Sema5A induces transient protein kinase C (PKC) phosphorylation of fascin-1, which can reduce its actin-binding/bundling activities and temporally parallels its translocation from cell body to extending processes. PKC inhibition or fascin-1 knockdown is sufficient to abrogate Sema5A-induced morphological differentiation, whereas the process is hastened by forced expression of fascin-1. Intriguingly, Sema5A induces re-expression of glial fibrillary acidic protein (GFAP), which when silenced restricts differentiation of glioma cells to bipolar instead of multipolar morphology. Therefore, we hypothesize complementary functions of fascin-1 and GFAP in the early and late phases of Sema5A-induced astrocytic differentiation of gliomas, respectively. In summary, Sema5A and plexin-B3 impede motility but promote differentiation of human gliomas. These effects are plausibly compromised in high-grade human astrocytomas in which Sema5A expression is markedly reduced, hence leading to infiltrative and anaplastic characteristics. This is evident by increased invasiveness of glioma cells when endogenous Sema5A is silenced. Therefore, Sema5A and plexin-B3 represent potential novel targets in counteracting glioma progression.

Evaluation of guggulipid and nimesulide on production of inflammatory mediators and GFAP expression in LPS stimulated rat astrocytoma, cell line (C6).

AIM OF THE STUDY: The present study was designed to investigate effect of guggulipid, a drug developed by CDRI and nimesulide on LPS stimulated neuroinflammatory changes in rat astrocytoma cell line (C6). MATERIALS AND METHODS: Rat astrocytoma cells (C6) were stimulated with LPS (10 microg/ml) alone and in combinations with different concentrations of guggulipid or nimesulide for 24h of incubation. Nitrite release in culture supernatant, ROS in cells, expressions of COX-2, GFAP and TNF-alpha in cell lysate were estimated. RESULTS: LPS (10 microg/ml) stimulated C6 cells to release nitrite, ROS generation, up regulated COX-2 and GFAP expressions at protein level and TNF-alpha at mRNA level. Both guggulipid and nimesulide significantly attenuated nitrite release, ROS generation and also down regulated expressions of COX-2, GFAP and TNF-alpha. Guggulipid and nimesulide per se did not have any significant effect on C6 cells. CONCLUSION: Results demonstrate the anti-inflammatory effect of guggulipid comparable to nimesulide which suggest potential use of guggulipid in neuroinflammation associated conditions in CNS disorders.

The role of leptin, soluble leptin receptor, resistin, and insulin secretory dynamics in the pathogenesis of hypothalamic obesity in children.

INTRODUCTION: In this study, we have investigated the role of leptin, soluble leptin receptor(sOb-R), resistin, and insulin secretory dynamics in the development of hypothalamic obesity. MATERIALS AND METHODS: Children who had hypothalamo-pituitary tumor were divided into two groups. First group included obese-overweight (hypothalamic obese = HOB group, n = 23) and second group included non-obese children (hypothalamic non-obese = HNOB group, n = 16). Exogenously obese-overweight children (OB group, n = 22) were included as controls. Basal and second-hour serum glucose and insulin in oral glucose tolerance test (OGTT), basal serum leptin, sOb-R, resistin levels, and homeostasis model assessment (HOMA) indexes were compared between the groups. RESULTS: Age, sex, and pubertal status were similar in study groups. Median and interquartile ranges of body mass index (BMI) z scores were similar in HOB and OB groups (2.0 (1.5-2.1) and 2.1 (1.8-2.3), respectively). Serum leptin levels corrected for BMI were highest and total leptin/sOb-R ratios (free leptin index (FLI)) tended to be higher in HOB than HNOB and OB groups, indicating leptin resistance (leptin/BMI, 4.0 (1.6-5.2), 1.5 (0.8-3.1), and 2.5 (1.8-3.5); FLI, 2.0 (0.8-3.5), 0.6 (0.3-1.2), and 1.5 (1-2.3) in HOB, HNOB, and OB groups; respectively). Serum resistin levels were similar in groups (2.6 (1.9-3.1), 2.8 (1.7-3.4), and 3.0 (2.2-3.5) ng/ml in HOB, HNOB, and OB groups, respectively). Basal serum glucose, basal and second-hour insulin levels in OGTT, and HOMA index were higher in OB group than the HOB and HNOB groups, indicating insulin resistance in simple obesity; however, increment of insulin to same glycemic load in OGTT was highest in the HOB group indicating insulin dysregulation (p < 0.05). CONCLUSION: Hypothalamic obesity seems to be related to both dysregulated afferent (leptin) and efferent (insulin) neural outputs through the autonomic nervous system resulting in energy storage as fat.

Inhibition of peroxide-induced radical generation by plant polyphenols in C6 astroglioma cells.

Plant polyphenols like flavonoids and hydroxystilbens have been found to possess radical scavenging/antioxidative activity, especially when studied in cell-free systems. A positive effect in such assays, however, does not necessarily indicate a protective activity against deleterious effects of oxidative stress in intact cells. In fact it has been shown that polyphenols can act as anti-oxidants as well as pro-oxidants. The aim of the present study was to investigate whether and with what potency selected polyphenols are able to inhibit cellular radical generation in C6 cells and whether they can induce oxidative stress themselves. Cumene hydroperoxide (CHP) was used as a model to induce radical generation which was measured by means of a fluorometric 2',7'-dichlorodihydro-fluorescein assay. CHP-induced, time and concentration dependent, a manifold increase of DCF fluorescence indicating intracellular radical generation. This process was inhibited by all the flavonoids and the hydroxystilben resveratrol, at low micromolar concentrations. The most potent compounds, luteolin and galangin, already at concentrations of 5 to 10 microM nearly completely abolished the radical generation in the presence of 500 microM CHP. The following ranking of anti-oxidative potency was obtained: luteolingalangin>kaempferol>quercetin>resveratrolgenisteintaxifolin. This ranking is completely different from that obtained by means of a trolox equivalent antioxidant capacity (TEAC) assay in a cell-free system, thus putting the biological relevance of the latter in question. Remarkably, one compound induced oxidative stress itself, namely genistein. This flavonoid inhibited the cellular radical generation in the presence of CHP while it significantly enhanced it in the absence of the peroxide.

Biological inhibitory effects of the Chinese herb danggui on brain astrocytoma.

OBJECTIVE: Previous studies have demonstrated the utility of the traditional Chinese herb danggui in the treatment of chronic myelogenous leukemia. Our aim was to examine whether it might similarly be used to treat glioblastoma multiforme. METHODS: The lipid-soluble active ingredients of danggui were extracted with acetone (AS-AC) or chlorophenol (AS-CH) and their antiproliferative and proapoptotic effects were studiedin vitro on cultured GBM 8401 cells and in vivoon tumors in nude mice. RESULTS: After a 24-hour treatment, either AS-AC or AS-CH at a lower (50 micro g/ml) and a higher concentration (100 micro g/ml) significantly inhibited the proliferative activity of GBM 8401 cultured cells by 30-50%, as well as the expression of cathepsin B and vascular endothelial growth factor (VEGF). In nude mice, the growth of the tumor was inhibited by 30% by AS-CH or AS-AC (20 mg/kg; p < 0.05) and by 60% by AS-CH or AS-AC (60 mg/kg; p < 0.05). AS-AC and AS-CH also significantly inhibited microvessel formation in the tumors of nude mice. CONCLUSIONS: Danggui may inhibit tumor growth by reducing the level of VEGF and the proapoptotic protein, cathepsin B. Thus, danggui may be useful in the treatment of high-grade astrocytomas.

In vivo research in astrocytoma cell proliferation with 1H-magnetic resonance spectroscopy: correlation with histopathology and immunohistochemistry.

INTRODUCTION: Assessment of brain tumor proliferative potential provides important prognostic information that supplements standard histopathologic grading. Proton magnetic resonance spectroscopy ((1)H-MRS) gives completely different information, relating to cell membrane proliferation, neuronal damage, energy metabolism and necrotic transformation of brain or tumor tissues. The aim of this study was to investigate the relationship between (1)H-MRS and tumor proliferative potential in astrocytomas. METHODS: We studied 34 patients with histologically verified astrocytomas using the (1)H-MRS protocol following routine MRI preoperatively. The tumor in 26 of these patients was classified as grade I/II (low grade), and the tumor in the remaining patients as grade III/IV (high grade) according to the World Health Organization classification criteria of nervous system tumors (2000). The tumor in 21 patients was homogeneous astrocytoma, and of these 17 were classified as low grade and 4 as high grade. Expression of proliferating cell nuclear antigen (PCNA) was determined immunohistochemically using streptavidin-biotin-peroxidase complex (SP) staining. RESULTS: The ratios of choline (Cho) to N-acetylaspartate (NAA) and Cho to creatine (Cr) in those with high-grade astrocytomas (n=4) were significantly higher than in those with low-grade astrocytomas (n=17) (t=2.899, P=0.009; t=3.96, P=0.001, respectively), and were found to be significantly correlated with the expression of PCNA in 21 patients with homogeneous astrocytomas (r=0.455, P=0.038; r=0.633, P=0.002, respectively). CONCLUSIONS: We conclude that (1)H-MRS may be a valuable method for predicting preoperatively the degree of malignancy of homogeneous astrocytomas by enabling the calculation of the Cho/NAA and Cho/Cr ratios in vivo, and indirect evaluation of the tumor proliferative potential and prognosis, which are not available using conventional magnetic resonance imaging (MRI).

A stochastic two-dimensional model of intercellular Ca2+ wave spread in glia.

We describe a two-dimensional stochastic model of intercellular Ca(2+) wave (ICW) spread in glia that includes contributions of external stimuli, ionotropic and metabotropic P2 receptors, exo- and ecto-nucleotidases, second messengers, and gap junctions. In this model, an initial stimulus evokes ATP and UTP release from a single cell. Agonists diffuse and are degraded both in bulk solution and at cell surfaces. Ca(2+) elevation in individual cells is determined by bound agonist concentrations s and by number and features of P2 receptors summed with that generated by IP(3) diffusing through gap junction channels. Variability of ICWs is provided by randomly distributing a predetermined density of cells in a rectangular grid and by randomly selecting within intervals values characterizing the extracellular compartment, individual cells, and interconnections with neighboring cells. Variability intervals were obtained from experiments on astrocytoma cells transfected to express individual P2 receptors and/or the gap junction protein connexin43. The simulation program (available as Supplementary Material) permits individual alteration of ICW components, allowing comparison of simulations with data from cells expressing connexin43 and/or various P2 receptor subtypes. Such modeling is expected to be useful for testing phenomenological hypotheses and in understanding consequences of alteration of system components under experimental or pathological conditions.

Inhibition of cellular proliferation and induction of apoptosis by curcumin in human malignant astrocytoma cell lines.

Nuclear factor (NF)-kappaB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-kappaB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-kappaB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-kappaB activity. In this study, we investigated inhibitory effects of curcumin on NF-kappaB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-kappaB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [(3)H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-kappaB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-kappaB activity by curcumin.

Differential activity of lipoic acid enantiomers in cell culture.

It is unclear whether the two enantiomeric forms (R & S) of lipoic acid (LA) share similar pharmacological activity and the exact cellular targets of LA are not well identified. We oxidatively stressed 3 cell culture systems representing different cell types. Mitochondrial metabolism was the primary endpoint. When C6 glioma was damaged by hydrogen peroxide (H2O2), all forms of LA protected. Racemic and S-LA were less effective than the R-isomer that was also protective in tertiary butyl hydroperoxide (TBHP)-damaged C6 glioma. In PC12 cells, little damage was produced by TBHP; R-LA increased mitochondrial metabolism above the level of non-damaged control. In H2O2 damaged PC12 cells, R-LA and racemic LA (but not S-LA) not only protected against damage, but increased mitochondrial metabolism above the non-damaged control level. When BAE cells were damaged with H2O2, R- and racemic LA protected while S-LA was ineffective.

Phospholipase C, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells.

We show that epigallocatechin-3 gallate (EGCG), a major component of green tea, stimulates phospholipase D (PLD) activity in U87 human astroglioma cells. EGCG-induced PLD activation was abolished by the phospholipase C (PLC) inhibitor and a lipase inactive PLC-gamma1 mutant, which is dependent on intracellular or extracellular Ca(2+), with the possible involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). EGCG induced translocation of PLC-gamma1 from the cytosol to the membrane and PLC-gamma1 interaction with PLD1. EGCG regulates the activity of PLD by modulating the redox state of the cells, and antioxidants reverse this effect. Moreover, EGCG-induced PLD activation was reduced by PKC inhibitors or down-regulation of PKC. Taken together, these results show that, in human astroglioma cells, EGCG regulates PLD activity via a signaling pathway involving changes in the redox state that stimulates a PLC-gamma1 [Ins(1,4,5)P(3)-Ca(2+)]-CaM kinase II-PLD pathway and a PLC-gamma1 (diacylglycerol)-PKC-PLD pathway.