Growth Of Astrocytoma Cells Research Articles

Correction: Inhibition of Furin-mediated Processing Results in Suppression of Astrocytoma Cell Growth and Invasiveness.

In the original version of [this article][1] ([1][2]), Figs. 1C and 3B are missing splice lines between U87MG and U118MG. The errors have been corrected in the latest online PDF version of the article. The authors regret this error. 1. 1.[↵][3]1. Mercapide J, 2. Lopez De Cicco R, 3

A simple method for fabrication of electrospun fibers with controlled degree of alignment having potential for nerve regeneration applications

In peripheral nerve injuries where direct suturing of nerve endings is not feasible, nerve regeneration has been facilitated through the use of artificially aligned fibrous scaffolds that provide directional growth of neurons to bridge the gap. The degree of fiber alignment is crucial and can impact the directionality of cells in a fibrous scaffold. While there have been multiple approaches that have been used for controlling fiber alignment, however, they have been associated with a compromised control on other properties, such as diameter, morphology, curvature, and topology of fibers. Therefore, the present study demonstrates a modified electrospinning set-up, that enabled fabrication of electrospun fibers with controlled degree of alignment from non-aligned (NA), moderately aligned (MA, 75%) to highly aligned (HA, 95%) sub-micron fibers while keeping other physical properties unchanged. The results demonstrate that the aligned fibers (MA and HA) facilitated directional growth of human astrocytoma cells (U373), wherein the aspect ratio of cells was found to increase with an increase in degree of fibers alignment. In contrast to NA and MA fibers, the HA fibers showed improved contact guidance to U373 cells that was demonstrated by a significantly higher cell aspect ratio and nuclear aspect ratio. In conclusion, the present study demonstrated a modified electrospinning setup to fabricate differentially aligned fibrous scaffolds with the HA fibers showing potential for use in neural tissue engineering.

Deguelins, Natural Product Modulators of NF1-Defective Astrocytoma Cell Growth Identified by High-Throughput Screening of Partially Purified Natural Product Extracts.

A high-throughput screening assay for modulators of Trp53/NF1 mutant astrocytoma cell growth was adapted for use with natural product extracts and applied to a novel collection of prefractionated/partially purified extracts. Screening 68 427 samples identified active fractions from 95 unique extracts, including the terrestrial plant Millettia ichthyotona. Only three of these extracts showed activity in the crude extract form, thus demonstrating the utility of a partial purification approach for natural product screening. The NF1 screening assay was used to guide purification of active compounds from the M. ichthyotona extract, which yielded the two rotenones deguelin (1) and dehydrodeguelin (2). The deguelins have been reported to affect growth of a number of cancer cell lines. They potently inhibited growth of only one of a panel of NF1/Trp53 mutant murine astrocytoma cell lines, possibly related to epigenetic factors, but had no effect on the growth of normal astrocytes. These results suggest the potential utility of deguelins as tools for further investigating NF1 astrocytoma cell growth. These bioprobes were identified only as a result of screening partially purified natural product extracts.

PM-10 * CDCA7L FUNCTIONS AS A MALE-SPECIFIC ONCOGENE IN ASTROCYTOMA

The most common types of primary brain tumors, astrocytoma and glioblastoma multiforme (GBM), are currently incurable. Both astrocytoma and GBM show male predominance, with a male to female ratio of 1.42:1 and 1.58:1, respectively. We performed linkage analysis in the Nf1-/ + ;Trp53-/ + cis (NPcis) mouse model of astrocytoma/GBM to identify a male-specific gliomagenesis modifier and further used combinatorial bioinformatics approaches as well as cross-species comparisons to prioritize male-specific candidate gene(s). Here, we showed that Cell division cycle-associated 7-like (CDCA7L), a myc cotranscription factor, is a male-specific oncogene in astrocytoma/GBM. CDCA7L expression is up-regulated in astrocytoma cells compared to normal brain, with males showing higher levels than females in both human and mouse. shRNA-mediated CDCA7L knockdown led to the decreased cell growth and viability of male-derived astrocytoma cells, but not female tumor cells in both human and mouse. Further mechanistic studies showed that CDCA7L depletion in male-derived astrocytoma cells led to the induction of cleaved Caspase-3 and p27 expression and reduction of Cyclin D1 expression. Furthermore, Cdca7l overexpression promotes the growth of WT mouse primary astrocytes only in males by inducing Cyclin D1 expression, but not in female astrocytes, suggesting a male-specific oncogenic role for CDCA7L in astrocytoma/GBM. Strikingly, CDCA7L depletion in human female U87MG GBM cells caused the increase of both growth and viability opposite to what is seen in male cells. Because male-female differences in CDCA7L action are hormone-independent, we examined whether the male-specific histone demethylase KDM5D regulates the effects of CDCA7L. We found that knockdown of KDM5D inhibits male-specific effects of CDCA7L on p27 and Cyclin D1 expression. Our data highlight the sex-specificity of CDCA7L in astrocytoma/GBM tumorigenesis and show that CDCA7L signaling pathways can be oncogenic in males, while being neutral or tumor suppressive in females. This has important implications for the application of therapies to human GBM patients.

Progesterone induces the growth and infiltration of human astrocytoma cells implanted in the cerebral cortex of the rat.

Progesterone (P4) promotes cell proliferation in several types of cancer, including brain tumors such as astrocytomas, the most common and aggressive primary intracerebral neoplasm in humans. In this work, we studied the effects of P4 and its intracellular receptor antagonist, RU486, on growth and infiltration of U373 cells derived from a human astrocytoma grade III, implanted in the motor cortex of adult male rats, using two treatment schemes. In the first one, fifteen days after cells implantation, rats were daily subcutaneously treated with vehicle (propylene glycol, 160 μL), P4 (1 mg), RU486 (5 mg), or P4 + RU486 (1 mg and 5 mg, resp.) for 21 days. In the second one, treatments started 8 weeks after cells implantation and lasted for 14 days. In both schemes we found that P4 significantly increased the tumor area as compared with the rest of the treatments, whereas RU486 blocked P4 effects. All rats treated with P4 showed tumor infiltration, while 28.6% and 42.9% of the animals treated with RU486 and P4 + RU486, respectively, presented it. Our data suggest that P4 promotes growth and migration of human astrocytoma cells implanted in the motor cortex of the rat through the interaction with its intracellular receptor.

The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone) is the major active agent of the alkaloid derivative of Camptotheca acuminata, with multiple pharmacological activities, including anticancer effects and promotion of apoptosis. The mechanism by which CIL-102 inhibits growth remains poorly understood in human astrocytoma cells. Herein, we investigated the molecular mechanisms by which CIL-102 affects the generation of reactive oxygen species (ROS) and cell cycle G2/M arrest in glioma cells. Treatment of U87 cells with 1.0μM CIL-102 resulted in phosphorylation of extracellular signal-related kinase (ERK1/2), downregulation of cell cycle-related proteins (cyclin A, cyclin B, cyclin D1, and cdk1), and phosphorylation of cdk1Tyr15 and Cdc25cSer216. Furthermore, treatment with the ERK1/2 inhibitor PD98059 abolished CIL-102-induced Cdc25cSer216 expression and reversed CIL-102-inhibited cdk1 activation. In addition, N-acetyl cysteine (NAC), an ROS scavenger, blocked cell cycle G2/M arrest and phosphorylation of ERK1/2 and Cdc25cSer216 in U87 cells. CIL-102-mediated ERK1/2 and ROS production, and cell cycle arrest were blocked by treatment with specific inhibitors. In conclusion, we have identified a novel CIL-102-inhibited proliferation in U87 cells by activating the ERK1/2 and Cdc25cSer216 cell cycle-related proteins and inducing ROS production; this might be a new mechanism in human astrocytoma cells.

Elevated Preoperative Glucose Levels and Survival in Elderly Newly Diagnosed Glioblastoma Patients (P07.111)

Objective: This retrospective study was undertaken to assess the relationship between preoperative glucose level and survival in elderly patients with newly diagnosed glioblastoma (GBM). Background Hyperglycemia has been associated with poor outcomes in many disease states. Furthermore, hyperglycemia augments in vitro astrocytoma cell growth. Design/Methods: We retrospectively reviewed 315 GBM patients, treated between July, 1998 and June, 2010. 18% (58/315) of the patients with known diabetes were excluded. The analysis was performed based on a recursive partitioning. Results: 242 GBM patients with a median age of 74 (range 65-91) were included in the final analysis. Among patients with performance status data (88%, 214/242) 23% had a KPS of 90-100 preoperatively, in 44% KPS was 70-80, and 34% had KPS scores 70, age 70, age 90 mg/dL), group three (KPS>70, age>75, any glucose level), and group four (KPS Conclusions: In our study, higher preoperative glucose level was associated with decreased overall survival in elderly patients diagnosed with GBM. Strict glucose control may contribute to improved outcome in treatment of these patients. Disclosure: Dr. Stevens has received personal compensation for activities with UCB Pharma. Dr. Ahluwalia has received personal compensation for activities with Elekta, Monteris Medical, and Merl.

Demonstration of vasculogenic mimicry in astrocytomas and effects of Endostar on U251 cells

Angiogenesis is an important process for the cell growth of normal and tumor tissues. Vasculogenic mimicry (VM) is a newly described vascular network structure that was first described in aggressive melanomas. To find out whether VM also exists in astrocytomas and to examine its clinical significance, we studied 80 malignant astrocytoma samples using immunohistochemistry coupled with periodic acid–Schiff (PAS) staining. To explore the possible therapeutic methods of anti-VM formation, we cultured astrocytoma cells using three-dimensional Matrigel and investigated the effects of Endostar, an endothelial cell growth inhibitor, on astrocytoma cell growth, invasion, and VM formation. VM structures were found in 8 samples of malignant astrocytomas, seven of which were grade IV astrocytomas. Glioblastoma U251 cells cultured in Matrigel formed vessel-like loops and networks, mimicking the features of VM in vivo, whereas such structures were not found in cultured normal astrocytes or well-differentiated astrocytoma SHG44 cells. In addition, treatment with Endostar led to a dose- and time-dependent inhibition of proliferation and invasion of both U251and SHG44 cells, but VM formation by U251 cells in vitro was not prominently affected. In conclusion, VM is frequently detected in aggressive glioblastomas, and the presence of VM may constitute a new predictor for poor prognosis in astrocytoma patients. Although Endostar has broad anti-tumor effects due to anti-angiogenesis and anti-tumor cell mechanisms, its inhibitory effects on VM formation by U251cells in vitro are not remarkable.

Targeting oncogenic miR-335 inhibits growth and invasion of malignant astrocytoma cells

BackgroundAstrocytomas are the most common and aggressive brain tumors characterized by their highly invasive growth. Gain of chromosome 7 with a hot spot at 7q32 appears to be the most prominent aberration in astrocytoma. Previously reports have shown that microRNA-335 (miR-335) resided on chromosome 7q32 is deregulated in many cancers; however, the biological function of miR-335 in astrocytoma has yet to be elucidated.ResultsWe report that miR-335 acts as a tumor promoter in conferring tumorigenic features such as growth and invasion on malignant astrocytoma. The miR-335 level is highly elevated in C6 astrocytoma cells and human malignant astrocytomas. Ectopic expression of miR-335 in C6 cells dramatically enhances cell viability, colony-forming ability and invasiveness. Conversely, delivery of antagonist specific for miR-335 (antagomir-335) to C6 cells results in growth arrest, cell apoptosis, invasion repression and marked regression of astrocytoma xenografts. Further investigation reveals that miR-335 targets disheveled-associated activator of morphogenesis 1(Daam1) at posttranscriptional level. Moreover, silencing of endogenous Daam1 (siDaam1) could mimic the oncogenic effects of miR-335 and reverse the growth arrest, proapoptotic and invasion repression effects induced by antagomir-335. Notably, the oncogenic effects of miR-335 and siDAAM1 together with anti-tumor effects of antagomir-335 are also confirmed in human astrocytoma U87-MG cells.ConclusionThese findings suggest an oncogenic role of miR-335 and shed new lights on the therapy of malignant astrocytomas by targeting miR-335.

Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.

Abstract 724: Selective β2-adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells

Abstract Selective β2-adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells Lawrence Toll1, Lucita Jimenez1, Willma Polgar1, Carol Green1, Jason Gow1, Kathleen O'Loughlin1, Michel Bernier2, Irving W. Wainer2 1 SRI International, Menlo Park, CA 2 Intramural Research Program, National Institute of Aging, Baltimore, MD We have reported the synthesis of fenoterol analogs and the characterization of these compounds as selective β2-adrenergic receptor (β2-AR) agonists, which have a broad range of abilities to stimulate cAMP accumulation in β2-AR transfected HEK cells, EC50cAMP. We now report the effect of a series of these compounds on the growth of 1321N1 astrocytoma cells (Table). In the 1321N1 cells, β2-AR stimulation by fenoterol analogs potently inhibited [3H]-thymidine incorporation with IC50 values that correlated with the EC50cAMP values. The effects were blocked by the β2-AR antagonist propranolol and mimicked by the adenylate cyclase activator forskolin. (R,R)-Fenoterol induced G1 arrest with an associated decrease in the proportion of cells in G2 and S phase and modulated the levels of proteins involved in cell division. The results indicate that selective β2-AR agonists may exert unanticipated antitumor effects against neural-derived neoplasms that express the β2-AR.Table.Functional activities of fenoterol analogs. Stimulation of cAMP accumulation was determined in intact HEK cells transfected with the β2-AR. Fenoterol analogs do not inhibit mitogenesis in HEK cells. Inhibition of [3H]-thymidine incorporation was conducted in 1321N1 cells in which the β2-AR is natively expressed. Results expressed as mean ± SD, n = 4.CompoundStimulation of cAMP AccumulationMitogenesis Inhibition EC50 (nM)% StimulationIC50 (nM)Isoproterenol0.20 ± 0.091000.05 ± 0.01(R,R)-fenoterol0.30 ± 0.09123.30 ± 8.790.14 ± 0.07(R,R)-4-methoxyfenoterol0.30 ± 0.23135.70 ± 11.030.17 ± 0.02(R,R)-ethylfenoterol2.80 ± 0.90125.40 ± 5.741.44 ± 0.27(R,R)-1-naphthylfenoterol12.5 ± 3.50129.20± 23.61.57 ± 0.34(R,R)-2-naphthylfenoterol0.40 ± 0.1290.30 ± 9.811.91 ± 0.57(R,R)-4-methoxy-1-naphthyl fenoterol3.90 ± 1.80105.90 ± 11.063.98 ± 0.28 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 724.

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

IGFBP2 Is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK

To identify targets critical to malignant childhood astrocytoma, we compared the expression of receptor tyrosine kinase- associated genes between low-grade and high-grade pediatric astrocytomas. The highest differentially overexpressed gene in high-grade astrocytoma is insulin-like growth factor- binding protein-2 (P = .0006). Immunohistochemistry confirmed overexpression of insulin-like growth factor-binding protein-2 protein (P = .027). Insulin-like growth factor- binding protein-2 stimulation had no effect on astrocytoma cell growth and migration, and minimally inhibited insulin-like growth factor-1-mediated migration, but not insulin-like growth factor-2-mediated migration. However, insulin-like growth factor-binding protein-2 stimulation significantly upregulated the major DNA repair enzyme gene, DNA-PKcs, and induced DNA-dependent protein kinase catalytic subunit protein expression in a time-dependent and dose-dependent manner, whereas insulin-like growth factor-1 had no effect. DNA-PKcs is also highly overexpressed by high-grade astrocytomas. These findings suggest insulin-like growth factor-binding protein-2 plays a role in astrocytoma progression by promoting DNA-damage repair and therapeutic resistance.

Autocrine regulation of glioblastoma cell-cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.

PERSISTENT OUTPATIENT HYPERGLYCEMIA IS INDEPENDENTLY ASSOCIATED WITH DECREASED SURVIVAL AFTER PRIMARY RESECTION OF MALIGNANT BRAIN ASTROCYTOMAS

Patients with malignant brain astrocytomas are at high risk for developing hyperglycemia secondary to frequent corticosteroid administration. Several clinical studies have shown that hyperglycemia is associated with poor outcome in multiple disease states. Furthermore, hyperglycemia augments in vitro astrocytoma growth, whereas hypoglycemia attenuates in vitro astrocytoma cell growth. We hypothesized that persistent hyperglycemic states in the outpatient setting may serve as a prognostic marker of decreased survival in patients with malignant brain astrocytomas. We retrospectively reviewed 367 cases of craniotomy for malignant brain astrocytomas (World Health Organization Grade III or IV). Persistent hyperglycemia was defined as serum glucose greater than 180 mg/dL occurring three or more times between 1 and 3 months postoperatively. Isolated hyperglycemia was defined as an isolated occurrence of serum glucose greater than 180 mg/dL. The independent association of outpatient glucose levels and recorded clinical and treatment variables with overall survival was assessed via multivariate proportional-hazards regression analysis. A total of 367 craniotomies (209 primary, 158 secondary) were performed for malignant brain astrocytomas (glioblastoma multiforme, 297; anaplastic astrocytomas, 70); 68 (19%) and 28 (8%) of the patients experienced isolated or persistent outpatient hyperglycemia, respectively. Patients experiencing persistent hyperglycemia were older (59 +/- 13 versus 51 +/- 14 yr), were diabetic more frequently (7 [25%] versus 10 [3%]), continued to receive corticosteroids more frequently (21 [75%] versus 35 [10%]); and received temozolomide less often (4 [14%] versus 116 [34%]). Adjusting for intergroup differences and variables associated with survival in this model, age (P = 0.001), Karnofsky Performance Scale score (P = 0.001), tumor grade (P = 0.001), primary versus secondary resection (P = 0.008), temozolomide (P = 0.007), subsequent resection (P = 0.07), and continued outpatient dexamethasone therapy, persistent outpatient hyperglycemia (relative risk, 1.79; 95% confidence interval, 1.05-3.05, P = 0.03) remained independently associated with decreased survival. Median survival for persistently hyperglycemic versus normal-glycemic cohorts was 5 and 11 months, respectively. In our experience, persistent outpatient hyperglycemia was associated with decreased survival in patients undergoing surgical resection for malignant astro- cytomas and was independent of the degree of disability, tumor grade, diabetes, prolonged dexamethasone use, or subsequent treatment modalities. Increased glucose control is warranted in this patient population and may contribute to improved outcomes in the treatment of malignant brain astrocytomas.

Matrix metalloproteinase‐9 interplays with the IGFBP2–IGFII complex to promote cell growth and motility in astrocytomas

Insulin-like growth factor II (IGFII) acts as a potent mitogen for several tumor types and has been reported to positively influence astrocytoma cell growth and motility. In the central nervous system, IGFII bioavailability is mainly modulated by insulin-like growth factor binding protein 2 (IGFBP2), which sequestrates IGFII and therefore prevents its interaction with the type-1 IGF receptor (IGF-IR). Proteolysis of IGFBP2 is the predominant mechanism recognized to reduce the binding affinity of IGFBP2 for IGFII, thus favoring dissociation of IGFII from the IGFBP2-IGFII complex. It is known that certain proteases involved in astrocytoma malignancy, such as matrix metalloproteinase-7 (MMP-7), plasmin, and cathepsin D, are able to proteolyze IGFBP2 in vitro. The present study aims to investigate whether other proteases expressed by astrocytomas, specifically MMP-2, MMP-9, and membrane-type 1 matrix metalloprotease (MT1-MMP), are able to proteolyze the IGFBP2-IGFII complex. Our results show the following: (i) MMP-9 proteolyzes the IGFBP2-IGFII complex in vitro, while MMP-2 and MT1-MMP do not; (ii) this MMP-9-induced IGFBP2-IGFII complex proteolysis releases free IGFII, which contributes to enhance the motility and the growth of LN229 astrocytoma cells. Furthermore, this study also highlights that the formation of the IGFBP2-IGFII complex inhibits IGFBP2's cell motility promoting effect by reducing the pool of free IGFBP2. In conclusion, MMP-9-induced IGFBP2 proteolysis may be regarded as an important post-translational event involved in astrocytoma aggressiveness. These new findings support drug targeting of MMP-9 as an interesting approach in the treatment of astrocytoma.

Epigenetic Silencing of the Protocadherin Family Member PCDH-γ-All in Astrocytomas

In a microarray-based methylation analysis of astrocytomas [World Health Organization (WHO) grade II], we identified a CpG island within the first exon of the protocadherin-γ subfamily A11 (PCDH-γ-A11) gene that showed hypermethylation compared to normal brain tissue. Bisulfite sequencing and combined bisulfite restriction analysis (COBRA) was performed to screen low- and high-grade astrocytomas for the methylation status of this CpG island. Hypermethylation was detected in 30 of 34 (88%) astrocytomas (WHO grades II and III), 20 of 23 (87%) glioblastomas (WHO grade IV), 8 of 8 (100%) glioma cell lines. There was a highly significant correlation (P = .00028) between PCDH-γ-A11 hypermethylation and decreased transcription as determined by competitive reverse transcription polymerase chain reaction in WHO grades II and III astrocytomas. After treatment of glioma cell lines with a demethylating agent, transcription of PCDH-γ-A11 was restored. In summary, we have identified PCDH-γ-A11 as a new target silenced epigenetically in astrocytic gliomas. The inactivation of this cell-cell contact molecule might be involved in the invasive growth of astrocytoma cells into normal brain parenchyma.

Effect of PSC 833, a potent inhibitor of P-glycoprotein, on the growth of astrocytoma cells in vitro.

Malignant astrocytomas have been found to express P-glycoprotein (Pgp, mdr1 gene product). It was hypothesized that in addition to conferring multidrug resistance, Pgp is intimately associated with the development of astrocytomas. Accordingly, we studied the effect of PSC 833 (PSC, Novartis), a potent inhibitor of Pgp, on the growth of Pgp-expressing astrocytoma cells. The results showed that in all the cell lines tested, PSC (10-60 microM) inhibited the growth as well as induced cell death. Cells exposed to PSC exhibited DNA ladder characteristic of apoptosis. PSC-induced cell death could be reversed by Z-VAD-fmk, a general caspase inhibitor, indicating that PSC-induced cell death was characteristic of caspase-mediated apoptosis. These results suggest a novel therapeutic strategy in the treatment of malignant astrocytomas by inhibitors of Pgp.

Influence of aspirin on growth and NOS expression in astrocytoma cell line

Objective To investigate the effect of Aspirin on the proliferation and NOS expression in astrocytoma cell line, and probe into its mechanism . Methods The effect of Aspirin on the growth of astrocytoma cells was evaluated by MTT assay; NOS protein levels were determined by immunohistochemistry, NO and CEA concentration in the medium were determined by Griess assay and lepton catch immunising method respectively.Results Aspirin can inhibit the growth of astrocytoma cells ,induce theexpressionofiNOS, increase the concentration of NO in the medium. The effect of these were in a concentration dependent pattern. Moreover,Aspirin can reduce the concentration of CEA in the medium. Conclusion Aspirin inhibit the growth of astrocytoma cell line . Up\|regulated iNOS expression resulting a increase of NO concentration are ascribed to mechanism of antiproliferation activity of Aspirin. CEA is a good indicator in monitoring curative effect of astrocytoma.

Identification of the Motif in Versican G3 Domain That Plays a Dominant-negative Effect on Astrocytoma Cell Proliferation through Inhibiting Versican Secretion and Binding

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.