Expression of tissue factor pathway inhibitor 2 inversely correlates during the progression of human gliomas.

Protease inhibitors regulate a variety of physiological and pathological processes including angiogenesis, embryo implantation, intravascular fibrinolysis, wound healing, and tumor invasion. Tissue factor pathway inhibitor (TFPI) 2 is a Mr 32,000 Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. In this study, we determined the relative amounts of TFPI-2 in low-, intermediate-, and high-grade human glioma cell lines and tumor tissue samples. TFPI-2 protein and mRNA levels (measured by Western and Northern blotting) were highest in low-grade glioma cells (Hs683), lower in anaplastic astrocytoma cells (SW1088 and SW1783), and undetectable in high-grade glioma cells (SNB19). Analysis of TFPI-2 protein in human normal brain and in glioma tumor tissues for TFPI-2 revealed the highest levels in normal brain, lesser amounts in low-grade gliomas and anaplastic astrocytomas, and undetectable amounts in glioblastomas. In situ hybridization of TFPI-2 mRNA with normal brain tissues revealed the greatest positivity in neurons, with moderate positivity in both glial and endothelial cells and moderate, little, or no TFPI-2 mRNA in low-grade glioma, anaplastic astrocytoma, and glioblastoma tumor tissue samples, respectively. We also found that recombinant TFPI-2 inhibited the invasiveness of SNB19 glioblastoma cells in a Matrigel assay in a dose-dependent manner. Collectively, these results suggest that TFPI-2 has a regulatory role in the invasiveness of gliomas in vitro and in vivo.     

A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa serine protease inhibitor found at high levels in extracellular matrix. Recombinant human TFPI-2 has recently been shown to be a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor XIa amidolytic activity. Earlier studies in our laboratory showed that the expression of TFPI-2 is lost during tumor progression in human gliomas. We stably transfected this protease inhibitor in multiform glioblastoma cell line (SNB-19) and in low-grade glioma cell line (Hs683) in sense and antisense orientation respectively. This confirmed that the upregulation/down-regulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas both in vitro and in vivo models. Collectively, these results suggested an idea to determine whether TFPI-2 is necessary for cell survival and inhibition of tumor formation in nude mice, due to apoptosis of intracerebrally injected SNB-19 cells. In the present study we determined p-ERK levels and found that they are decreased in TFPI-2 over-expressed clones (SNB-19) and increased in TFPI-2 down-regulated clones (Hs683). We also checked the levels of BAX/BCl-2, caspases (for e.g., 9, 7, 3, 8), PARP, cytochrome-c and Apaf-1. Moreover, the increase of apoptosis in vitro is associated with increased and decreased expression of apoptotic protein BAX in sense clones (SNB-19) and antisense clones (Hs683) respectively, when compared to controls and vice versa with Bcl-2 the anti-apoptotic protein. Caspases (9, 7 and 3), cytochrome-c, Apaf-1 and PARP levels are increased in SNB-19 and decreased in Hs683. Caspase 8 was not expressed in either cell line. Caspases 9 and 3 activity assay revealed higher activity in sense clones (SNB-19) but lesser in antisense clones (Hs683) compared to controls. This is the first report of TFPI-2 playing a novel role in cell survival in human gliomas.     

Temozolomide-induced modification of the CXC chemokine network in experimental gliomas

CXCL chemokines display important roles in glioblastoma (GBM) biology, including cell proliferation, death and migration features. While temozolomide (TMZ) represents the standard chemotherapeutic used to treat GBM patients, its role in CXCL networking in GBMs remains unexplored. The effects of short-term and long-term in?vitro treatment with temozolomide on CXCL chemokine expression were characterized in human malignant glioma cell lines. U373 and T98G astroglioma and Hs683 oligodendroglioma cells were cultured for months in the presence of increasing concentrations of TMZ (up to 1 mM), and their whole genome profiles were analyzed along with a complete mapping of all CXCL chemokines and their respective receptor mRNAs. The study was extended to an additional established cell line and four primocultures. The in vitro results were compared with a clinical series of 156 human gliomas and 23 normal brain tissue samples. The expression and secretion of CXCL2, CXCL3 and CXCL8 following different TMZ treatments were determined in Hs683, U373 and T98G glioma cells. The long-term TMZ-treated astroglioma cells, but not the Hs683 oligodendroglioma cells, developed in vivo a certain level of resistance to TMZ, which correlated with the up- regulation of CXCL2, CXCL3 and CXCL8 expression in the U373 and T98G astroglioma cells. The transient down-regulation of CXCL2 in Hs683 glioma cells using siRNA markedly impaired their proliferation rate. In conclusion, TMZ affects the expression and secretion of CXCL2 (and, to a lesser extent, CXCL3 and CXCL8) in glioma cells, and CXCL2 directly impacts glioma cell biology.     

Evaluation of the efficiency of chemotherapy in in vivo orthotopic models of human glioma cells with and without 1p19q deletions and in C6 rat orthotopic allografts serving for the evaluation of surgery combined with chemotherapy

BACKGROUND: Malignant gliomas of the central nervous system remain associated with dismal prognoses because of their diffuse invasion of the brain parenchyma. Very few experimental models that mimic clinical reality are available today to test potentially new therapies. The authors set up experimental in vivo glioma models of anaplastic astrocytomas of human and rat origins and anaplastic oligodendroglioma of human origin. Standard hospital chemotherapies were employed to test the validity of these models. METHODS: Three glioma cells lines obtained from the American Type Culture Collection (i.e., human Hs683 and U373 cells and rat C6 cells) were implanted into nude mouse brains (Hs683 and U373 cells) and rat brains (C6 cells). The astrocytic nature, as opposed to the oligodendrocytic nature, of the Hs683 and U373 models was investigated by using quantitative (computer-assisted microscopy) immunohistochemical characterizations of nestin, vimentin, glutathione-S-transferase alpha (GSTalpha), GSTmu, GSTpi, and p53 expression. Comparative genomic hybridization (CGH) was employed to investigate 1p19q losses. Chronic administrations of carmustine (BCNU), fotemustin, or temozolomide were assayed in the xenografted U373 and Hs683 models. Both BCNU-related chemotherapy and surgery were assayed in the C6 model. RESULTS: The quantitative phenotypic analyses pointed to the oligodendroglial nature of the Hs683 cell line and the astrocytic nature of the U373 cell line. The Hs683 cells exhibited 1p19q losses, whereas the U373 cells did not. BCNU, fotemustin, and temozolomide dramatically increased the time of survival of the Hs683 oligodendroglioma-bearing mice, whereas temozolomide only induced a weak but nevertheless statistically significant increase in the U373 glioma-bearing mice. In the C6 rat glioma model, surgery and BCNU chemotherapy were more efficient than either treatment alone. CONCLUSIONS: The in vivo models of gliomas of the central nervous system developed in the current work best mimicked clinical reality. They can be used either to identify new therapies against human gliomas or to optimize existing therapies.     

Galectin 1 Proangiogenic and Promigratory Effects in the Hs683 Oligodendroglioma Model Are Partly Mediated through the Control of BEX2 Expression

We have previously reported that galectin 1 (Gal-1) plays important biological roles in astroglial as well as in oligodendroglial cancer cells. As an oligodendroglioma model, we make use of the Hs683 cell line that has been previously extensively characterized at cell biology, molecular biology, and genetic levels. Galectin 1 has been shown to be involved in Hs683 oligodendroglioma chemoresistance, neoangiogenesis, and migration. Down-regulating Gal-1 expression in Hs683 cells through targeted small interfering RNA provokes a marked decrease in the expression of the brain-expressed X-linked gene: BEX2. Accordingly, the potential role of BEX2 in Hs683 oligodendroglioma cell biology has been investigated. The data presented here reveal that decreasing BEX2 expression in Hs683 cells increases the survival of Hs683 orthotopic xenograft-bearing mice. Furthermore, this decrease in BEX2 expression impairs vasculogenic mimicry channel formation in vitro and angiogenesis in vivo, and modulates glioma cell adhesion and invasive features through the modification of several genes previously reported to play a role in cancer cell migration, including MAP2, plexin C1, SWAP70, and integrin β₆. We thus conclude that BEX2 is implicated in oligodendroglioma biology.     

Metastasis tumor-associated protein-2 knockdown suppresses the proliferation and invasion of human glioma cells in vitro and in vivo

Metastasis tumor-associated protein 2 (MTA2) is a member of the MTA family that is closely associated with tumor progression and metastasis. However, the role of MTA2 in glioma cells remains unclear. The expression of MTA2 was measured using immunohistochemistry and western blotting in the human brain tumor tissue array and human glioma cell lines. The impact of MTA2 knockdown on GBM8401 and Hs683 cell growth was evaluated by MTT assay and flow cytometry. Cell migration and invasion were analyzed by cell-migration assay and Matrigel invasion assay. In addition, we used subcutaneous tumor models to study the effect of MTA2 on the growth of glioma cells in vivo. We found that MTA2 protein and mRNA expression are higher in GBM8401 and Hs683 cells than in other glioma cells (M059 J, M059 K and U-87 MG), and glioma tumor tissue correlated significantly with tumor grade (P < 0.001). Knockdown of MTA2 expression significantly inhibited cell growth, cell migration and invasion, and induced G0/G1 phase arrest in human GBM8401 and Hs683 cells in vitro. Moreover, in vivo studies using subcutaneous xenografts in mice models indicate that MTA2 knockdown significantly inhibited tumorigenicity. These results indicate that MTA2 plays an important oncogenic role in the development and progression of gliomas.     

EphA2和MMP2在胶质瘤组织表达及其相关性

[目的]探讨人脑胶质瘤组织中EphA2和MMP2的表达水平及相关性。[方法]采用免疫组织化学SP法检测69例胶质瘤组织、10例正常脑组织中EphA2和MMP2蛋白表达情况,分析两者表达水平与胶质瘤临床病理分级之间的关系及两者的相关性。[结果]正常脑组织、低级别胶质瘤组织（Ⅰ～Ⅱ级）和高级别胶质瘤组织（Ⅲ～Ⅳ级）中EphA2蛋白表达的阳性率分别为10.0%（1/10）、37.0%（10/27）、90.5%（38/42）,差异有统计学意义（χ2=33.0826,P=0.000）;正常脑组织、低级别胶质瘤组织和高级别胶质瘤组织中MMP2蛋白表达的阳性率分别为0、14.8%（4/27）、69.0%（29/42）,差异有统计学意义（χ2=28.0869,P=0.000）。在胶质瘤组织中,EphA2与MMP2蛋白表达呈正相关（r=0.660,P=0.000）。[结论]EphA2与MMP2同时高表达于胶质瘤组织中,EphA2可能通过影响MMP2而促进肿瘤的侵袭与转移。     

Long-term Temozolomide Treatment Induces Marked Amino Metabolism Modifications and an Increase in TMZ Sensitivity in Hs683 Oligodendroglioma Cells

Gliomas account for more than 50% of all primary brain tumors. The worst prognosis is associated with gliomas of astrocytic origin, whereas gliomas with an oligodendroglial origin offer higher sensitivity to chemotherapy, especially when oligodendroglioma cells display 1p19q deletions. Temozolomide (TMZ) provides therapeutic benefits and is commonly used with radiotherapy in highly malignant astrocytic tumors, including glioblastomas. The actual benefits of TMZ during long-term treatment in oligodendroglioma patients have not yet been clearly defined. In this study, we have investigated the effects of such a long-term TMZ treatment in the unique Hs683 oligodendroglioma model. We have observed increased TMZ sensitivity of Hs683 orthotopic tumors that were previously treated in vitro with months of progressive exposure to increasing TMZ concentrations before being xenografted into the brains of immunocompromised mice. Whole-genome and proteomic analyses have revealed that this increased TMZ sensitivity of Hs683 oligodendroglioma cells previously treated for long periods with TMZ can be explained, at least partly, by a TMZ-induced p38-dependant dormancy state, which in turn resulted in changes in amino acid metabolism balance, in growth delay, and in a decrease in Hs683 oligodendroglioma cell-invasive properties. Thus, long-term TMZ treatment seems beneficial in this Hs683 oligodendroglioma model, which revealed itself unable to develop resistance against TMZ.     

人胚肾细胞HEK293中miRNA-128-1表达调控机制的初步研究

目的:初步研究人胚肾细胞HEK293中miRNA-128-1的表达调控机制,为进一步探讨miRNA-128-1在胶质瘤中的表达调控奠定基础。方法:通过软件UCSC预测miRNA-128-1的启动子区域;运用软件UCSC、CBIL预测可能与miRNA-128-1启动子区域结合的候选转录因子;构建相关质粒,包括pB-miRNA-128-1、pShNF1、pShc-Myc、pShTAF1、pShYY1、pShMAF、pShRELA2、pShRNUX1和pShNFkappaB。然后将pB-miRNA-128-1分别与上述质粒共转染人胚肾细胞HEK293,检测报告基因活性,确定候选转录因子的作用性质。结果:共转染pShc-Myc可显著抑制pB-miRNA-128-1启动子报告基因的活性;共转染pShNFkappaB可显著升高pB-miRNA-128-1启动子报告基因的活性;共转染pShNF1、pShTAF1、pSbYY1、pShMAF、pShRELA2或pShRNUX1对pB-miRNA-128-1启动子报告基因的活性未见明显影响。结论:在HEK293细胞miRNA-128-1的表达调控中,c-Myc可能发挥转录激活作用,而NFkappaB可能发挥转录抑制作用。     

Transcriptional upregulation of SPARC,in response to c-Jun overexpression,contributes to increased motility and invasion of MCF7 breast cancer cells

Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotype changes related to malignant progression including increased motility and invasion. Concurrent with these phenotypic effects are changes in the expression of multiple gene targets. We previously demonstrated that expression of the SPARC/osteonectin gene, while undetectable in the MCF7 cell line, is highly induced in response to stable c-Jun overexpression (c-Jun/MCF7). Because the SPARC gene product is associated with tumor cell invasion in a variety of different cancers, we have examined its role in mediating the phenotypic changes induced by c-Jun in MCF7 cells. We found that antisense mediated suppression of SPARC dramatically inhibits both motility and invasion in this c-Jun/MCF7 model. In contrast, stable overexpression of SPARC in the parental MCF7 cell line is not sufficient to stimulate cell motility or invasion. Examination of the promoter region of the human SPARC gene reveals three non-canonical AP-1 sites. We demonstrate that one of these sites binds c-Jun/Fra1 heterodimers in vitro, but that this and the other AP-1 like sites are dispensable with respect to c-Jun stimulated SPARC promoter activation. Deletion analysis identified a region between -120 and -70 as a c-Jun responsive element sufficient to induce maximal promoter activation. This region does not contain any AP-1 sites but does mediate binding by SP1 'like' complexes. Furthermore, this region is necessary for SP1/SP3 responsiveness in Drosophila SL2 cells. These results demonstrate that SPARC plays an important role in stimulating motility and the invasive behavior of c-Jun/MCF7 cells and that SPARC promoter activation by c-Jun appears to occur through an indirect mechanism.     

The role of JNK and p38 MAPK activities in UVA-induced signaling pathways leading to AP-1 activation and c-Fos expression

To further delineate ultraviolet A (UVA) signaling pathways in the human keratinocyte cell line HaCaT, we examined the potential role of mitogen-activated protein kinases (MAPKs) in UVA-induced activator protein-1 (AP-1) transactivation and c-Fos expression. UVA-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK) proteins was detected immediately after irradiation and disappeared after approximately 2 hours. Conversely, phosphorylation of extracellular signal-regulated kinase was significantly inhibited for up to 1 hour post-UVA irradiation. To examine the role of p38 and JNK MAPKs in UVA-induced AP-1 and c-fos transactivations, the selective pharmacologic MAPK inhibitors, SB202190 (p38 inhibitor) and SP600125 (JNK inhibitor), were used to independently treat stably transfected HaCaT cells in luciferase reporter assays. Both SB202190 and SP600125 dose-dependently inhibited UVA-induced AP-1 and c-fos transactivations. SB202190 (0.25-0.5 microM) and SP600125 (62-125 nM) treatments also primarily inhibited UVA-induced c-Fos expression. These results demonstrated that activation of both JNK and p38 play critical role in UVA-mediated AP-1 transactivation and c-Fos expression in these human keratinocyte cells. Targeted inhibition of these MAPKs with their selective pharmacologic inhibitors may be effective chemopreventive strategies for UVA-induced nonmelanoma skin cancer.