The Effect of Hyaluronic Acid on the Invasiveness of Malignant Glioma Cells : Comparison of Invasion Potential at Hyaluronic Acid Hydrogel and Matrigel

Objective

Hyaluronidase (HAse), a degrading enzyme of hyaluronic acid (HA), is highly expressed in patients with malignant glioma. The purpose of this study was to verify whether HAse is related to the invasion of glioma cells. We also investigated if glioma cells with higher mobility in 2-dimensioal (2-D) method have also higher mobility at 3-dimensional (3-D) environment.

Methods

Malignant glioma cell lines (U87MG, U251MG, U343MG-A, and U373MG) were used, and their HAse expressions were evaluated by HA zymography. The migration ability was evaluated by simple scratch technique. The invasiveness of each cell lines was evaluated by Matrigel invasion assay and HA hydrogel invasion assay. In HA hydrogel invasion assay, colonies larger than 150 µm were regarded as positive ones and counted. Statistical analysis of migration ability and invasion properties of each cell lines was performed using t-test.

Results

In scratch test to examine migration ability of each cell lines, U87MG cells were most motile than others, and U343MG-A least motile. The HAse was expressed in U251MG and U343MG-A cell lines. However, U87MG and U373MG cell lines did not express HAse activity. In Matrigel invasion assay, the cell lines expressing HAse (U251MG and U343MG-A) were more invasive in the presence of HA than HAse deficient cell lines (U87MG and U373MG). In HA hydrogel invasion assay, the HAse-expressing cell lines formed colonies more invasively than HAse-deficient ones.

Conclusion

Malignant Glioma cells expressing HAse were more invasive than HAse-deficient ones in 3-dimensional environment. Therefore, it might be suggested that invasion of malignant gliomas is suppressed by inhibition of HAse expression or HA secretion. Additionally, the ability of 2-D migration and 3-D invasion might not be always coincident to each other in malignant glioma cells.     

Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

Caveolin-1 expression is maintained in rat and human astroglioma cell lines

Caveolin-1 is the principal structural and functional component of caveolae, a plasmalemmal compartment that has been proposed to sequester lipid and protein components that participate in transmembrane signal transduction processes. Multiple studies reveal a reduction in the expression level of caveolin-1 mRNA and protein in many carcinomas as well as transformed cells. The human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). Collectively, these data have been taken to imply that caveolin-1 may function in a tumor suppressor capacity. To determine if a reduction in the expression level of caveolin-1 mRNA and protein accompanied the transformation of astrocytes, we undertook studies of two transformed rat astroglial cell lines, C6 and DI TNC(1), as well as several cell lines derived from human glioblastoma tumors: T98G, U87MG, U118MG, U138MG, and U373MG. Ultrastructural, immunolocalization, immunoblot, and Northern blot analyses demonstrated that caveolin-1 message and protein were expressed in all rat and human glioma cells. The localization pattern, buoyant density, and detergent-insolubility property of caveolin-1 protein were indistinguishable from that determined for nontransformed type 1 astrocytes in culture. Nucleotide sequence analyses of caveolin-1 cDNAs indicate that mutations are not present in the caveolin-1 sequence in any of the glioma cell types. Taken together with previous analyses, these data indicate that, at least for astrocytes, the process of transformation in and of itself is not solely sufficient to reduce the level of caveolin-1 expression, and that caveolin-1 expression in and of itself is not solely sufficient to prevent the acquisition of a transformed phenotype.     

High concentrations of cannabinoids activate apoptosis in human U373MG glioma cells

Cannabinoids bind to two G-protein-coupled receptors, CB1 and CB2, expressed by neurons and cells of the immune system, respectively. Glioma cells (astrocyte-derived brain tumor cells) express cannabinoid receptors, and numerous in vitro and in vivo studies performed in rodents have concluded that apoptosis could be induced by cannabinoids in these cells. Whether this also applies to human cells is controversial; we, therefore, assessed the effect of cannabinoids on human glioma cell viability with the human astrocytoma cell line U373MG. We report here that U373MG human glioma cells are sensitive only to high concentrations of cannabinoids (>5 microg/ml for Delta(9)-THC). Similar concentrations of the compounds promoted a rapid activation of extracellular-regulated kinase and c-Jun NH2-terminal kinase, suggesting that cannabinoid receptors are functional in U373MG cells. Nevertheless, these kinases are not involved in cannabinoid-induced cell death in U373MG cells, insofar as blocking their activation with specific inhibitors does not reduce cell death. CB1 is expressed in U373MG cells and is involved in cannabinoid-induced cell death, in that blocking its activation with a specific antagonist (AM251) almost totally prevented cell death following incubation of the cells with Delta(9)-THC. In addition, as already reported, some cannabinoids may have modest proproliferative properties in U373MG cells. Human U373MG glioma cells are sensitive only to very high, pharmacologically irrelevant concentrations of cannabinoids, so it seems unlikely that cannabinoids would constitute promising molecules for treating malignant astrocytoma; they do not induce glioma cell death at doses that could be applied safely to humans.     

表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化

目的 探讨表达人血管生成抑制因子１（VASH1）的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法 构建针对VASH1的慢病毒载体pGCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 mRNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果 成功构建pGCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70％以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 mRNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低（P<0.01）,而且U-87MG细胞凋亡率明显增加（P<0.01）。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。     

Role of high molecular weight extracellular matrix proteins in glioma cell migration

Malignant human gliomas are characterized by an uncontrolled cell proliferation and infiltrative growth within the brain. Complete surgical removal is difficult due to disseminated tumour cells, and the fundamental mechanisms responsible for this spread are poorly understood. An extensive tumour cell movement along blood vessels is frequently observed and this may be due to specific interactions between tumour cell surface receptors and specific extracellular matrix (ECM) components present in conjunction with vascular elements. In order to investigate the influence of ECM on glioma cell migration, three different human glioma cell lines (U-373 MG, A-172 MG and HF-66) were exposed to known ECM components of the basement membrane (laminin, fibronectin and collagen type IV). Cell migration from multicellular spheroids was studied, using a custom-made medium which was prepared by removing the high molecular weight protein fraction (>100 kDa) from newborn calf serum by ultrafiltration. To this medium, the specific ECM components were added. For two of the cell lines (A-172 MG and U-373 MG), laminin was the most potent stimulator of glioma cell migration; the effect of laminin exceeded that evoked by ordinary serum-supplemented medium. For the HF-66 cell line, fibronectin was the most potent stimulator of migration. Western blot analysis showed that the A-172 MG and HF-66 cell lines expressed low amounts of laminin compared with U-373 MG, which showed extensive intrinsic synthesis of this ligand. U-373 MG was the only cell line that migrated in pure filtered medium. The cells stimulated by fibronectin expressed a different morphology from those stimulated by laminin suggesting that specific ECM-receptor binding may activate different cytoskeletal components within the cells. Furthermore, it was shown that there was no difference in the amount of protein synthesis between cells grown in filtered medium and in filtered medium supplemented with different ECM components. This suggests that ECM-induced cell migration is not dependent on a high level of protein synthesis. It is also shown that α3 integrin, which is a receptor-subunit for laminin, fibronectin and collagen type IV, was highly expressed in all cell lines. This study indicates that glioma cells need serum proteins with a molecular weight >100 kDa to migrate in vitro, and that laminin and fibronectin play an important role in this process.     

Differential control of VEGF synthesis and secretion in human glioma cells by IL-1 and EGF.

VEGF (vascular endothelial growth factor), one of the most potent angiogenic factors, has recently been identified as an inducer of neoangiogenesis in many tumors including gliomas. VEGF itself appears to be regulated through different pathways. Since malignant gliomas frequently show EGF receptor amplification and express IL-1, a pivotal regulatory cytokine involved in angiogenesis, we analyzed interactions between EGF/EGF receptor and IL-1/IL-1 receptor and VEGF in the established glioblastoma cell lines U-87 MG and A-172. Basal VEGF expression was an order of magnitude higher in U-87 MG compared to A-172. IL-1 caused a fast and strong increase of VEGF secretion in U-87 MG which appeared to harbor an intracellular VEGF pool for enhanced exocytosis. The IL-1 receptor antagonist (IL-1-ra) reversed this effect suggesting an IL-1 receptor-associated mechanism. In contrast, VEGF secretion could not be increased by exogenous IL-1 exposure in A-172, which apparently lacked an intracellular VEGF pool for augmented exocytosis. However, IL-1-ra treatment alone caused a significant reduction of basal VEGF secretion in both U-87 MG and A-172. This suggests that baseline secretion of VEGF involves IL-1 receptor activation by endogenously produced IL-1. EGF also stimulated the secretion of VEGF into the cell supernatant. However, this effect, observed in both U-87 MG and A-172, was delayed and only occurred following replenishment of the intracellular VEGF pool. EGF upregulated the amount of VEGF mRNA. In general, the effects of IL-1 and EGF on VEGF were additive, suggesting independent mechanisms. Since IL-1 appears to be involved in VEGF secretion in glial tumors through an autocrine/paracrine mechanism, recombinant human IL-1-ra may evolve as a new agent for anti-angiogenic glioma therapy.     

Knockdown of annexin 2 decreases migration of human glioma cells in vitro

Diffuse invasion of brain tissue is a major reason for the poor prognosis of patients with glioblastoma. Annexin 2, a member of the large annexin family of Ca2+ and membrane-binding proteins, is expressed at high protein levels in human gliomas and has been proposed as a marker of glioma malignancy, while its functional role in these tumours is unknown so far. The ability of annexin 2 to interact with the actin cytoskeleton, as well as its potential to bind invasion-associated proteases, suggests that it could participate in invasion-associated processes in human gliomas. Therefore, we analysed here functional consequences of RNA interference-mediated silencing of annexin 2 in U87MG and U373MG human glioma cell lines. While no impact of annexin 2 downregulation on proliferation and adhesion was observed, our analyses revealed that migration of U87MG and U373MG cells was significantly inhibited following annexin 2 depletion. This effect was not related to a compensatory increase of the related annexins 1 or 6. Our findings identify annexin 2 as a potential candidate involved in glioma invasion and support the potential of RNA interference as powerful tool in the decryption of glioma invasion mechanisms.     

九节龙皂苷对胶质瘤U373MG的抑制作用及其机制研究

目的 探讨九节龙皂苷对人脑胶质瘤细胞U373MG增殖的抑制作用及其机制.方法 培养U373MG细胞系,随机分为二甲基亚砜对照组(control组)和九节龙皂苷治疗(ADS)组.采用甲基噻唑基四唑法检测不同剂量ADS对人胶质瘤细胞U373MG增殖的影响;流式细胞仪观察细胞凋亡情况.免疫组化检测凋亡蛋白Caspase-3、p-Caspase-3的表达变化.结果 与对照组比较,ADS可显著降低U373MG细胞的生存率;流式细胞仪检测结果显示,随着ADS浓度的增大,U373MG细胞的凋亡率明显上升,免疫组化结果同样证明,ADS可呈剂量依赖性的促进凋亡信号Caspase-3和p-Caspase-3的高表达,不同组间差异有统计学意义(P＜0.05).结论 ADS可呈剂量依赖性的抑制U373MG细胞增殖,可能通过促进Caspase-3、p-Caspase-3的表达,诱发U373 MG细胞大量凋亡,发挥重要的抗肿瘤作用.     

Differential expression of glial fibrillary acidic protein in human glioma cell lines

Summary We have obtained a cDNA fragment to human glial fibrillary acidic protein (GFAP) by immunoscreening a gt11 human brain cDNA library with antibody to bovine GFAP. The highly homologous nucleotide sequence of this clone with that of the mouse GFAP enabled the identification of this cDNA as one encoding GFAP. As this cDNA hybridized with a single major RNA species in Northern blots of RNA from human and mouse brain tissues and gave one or two bands in Southern blots of human genomic DNA, it was considered to be specific for GFAP. Using this cDNA as a probe we investigated the levels of GFAP expression in ten human glioma cell lines. A 3.5-kb GFAP mRNA was detected in five of the ten glioma cell lines, one of which was U-251 MG cell line and the other four were clones derived from the same tumor (CL1, 2, 3, and 4). There was a difference in the amount of GFAP mRNA among U-251 MG and the four clonal cell lines. Quantitative evaluation of this difference by RNA dot blot analysis revealed that the amount of GFAP mRNA expressed in CL3 was about 1/5 and in CL4 about 1/10 the amount expressed in U-251 MG, CL1, and CL2. Semiquantitative Western blot analysis showed that GFAP levels corresponded to the GFAP mRNA levels in these cell lines. By Southern blot analysis of genomic DNA the GFAP gene was similarly detected in all of these cell lines regardless of the level of GFAP expression. Thus, by using a cDNa to human GFAP we have demonstrated the presence of clonal cell lines from human glioma showing different levels of GFAP expression, which may provide a useful basis for further investigations on the regulation of GFAP gene expression in glial cells.     

β‐Catenin expression in human neural cell lines following exposure to cytokines and growth factors

β‐Catenin acts as a key mediator of the Wnt/Wingless signaling pathway involved in cell proliferation, differentiation and survival. Recent studies have shown that an unstable interaction between β‐catenin and the mutant presenilin‐1 induces neuronal apoptosis, and that β‐catenin levels are decreased in the brains of patients with Alzheimer’s disease (AD). Since activated microglia and astrocytes play a role in the process of neuronal degeneration in AD, the cytokine/growth factor‐regulated expression of β‐catenin in human neural cell lines, including NTera2 teratocarcinoma‐derived differentiated neurons (NTera2‐N), IMR‐32 neuroblastoma, SKN‐SH neuroblastoma and U‐373MG astrocytoma, was studied quantitatively following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain‐derived neurotrophic factor (BDNF), tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, IL‐6, interferon (IFN)‐γ, transforming growth factor (TGF)‐β1, dibutyryl cyclic adenosine 3′,5′‐cyclic monophosphate (cAMP) (dbcAMP) or phorbol 12‐myristate 13‐acetate (PMA). β‐Catenin mRNA expressed constitutively in all of these cell lines was unaffected by treatment with any factors examined. In contrast, β‐catenin protein levels were reduced markedly in NTera2‐N cells by exposure to dbcAMP, EGF or bFGF, and in U‐373MG cells by treatment with dbcAMP or PMA, but were unaffected in any cell lines by BDNF, TNF‐α, IL‐1β, IL‐6, IFN‐γ or TGF‐β1. These results indicate that β‐catenin is expressed constitutively in human neural cells and downregulated at a protein level by a set of growth factors in a cell type‐specific manner.     

Constitutive and cytokine-regulated expression of presenilin-1 and presenilin-2 genes in human neural cell lines

To investigate the role of pleiotropic neuronal and glial cytokines in the regulation of presenilin (PS) gene expression in human neural cells, both presenilin-1 (PS1) and presenilin-2 (PS2) mRNA levels were analysed by Northern blotting in SK-N-SH neuroblastoma, IMR-32 neuroblastoma, NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N) and U-373MG astrocytoma cells following exposure to proinflammatory cytokines (TNF-alpha, IFN-gamma, or IL-1beta), anti-inflammatory cytokines (IL-10 or TGF-beta1), dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate (PMA). The constitutive expression of PS1 (3.0 kb) and PS2 (2.3 kb) mRNA was identified in all these cell lines, in which PS1 mRNA levels were unaltered following treatment with any cytokines and factors examined. By contrast, PS2 mRNA expression was upregulated substantially in SK-N-SH cells by exposure to TNF-alpha and in U-373MG cells by treatment with IFN-gamma, whereas it was downregulated in both NTera2-N and U-373 MG cells following exposure to IL-1beta or PMA. The levels of PS2 mRNA remained unchanged in IMR-32 cells after these treatments. These results indicate that PS1 and PS2 genes are expressed constitutively in a panel of human neural cell lines where PS2 mRNA expression is affected by a distinct set of cytokines via cell type-specific mechanisms that do not alter PS1 mRNA levels, suggesting the existence of separated regulatory systems controlling the expression of PS1 and PS2 genes in human neural cells.     

Modulation by tumor necrosis factor-alpha of human astroglial cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF).

Phagocyte survival and function are enhanced by GM-CSF and G-CSF. The production of both CSFs can be induced in mesenchymal cells by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). We have recently demonstrated that IL-1 alpha and beta induced the production of GM-CSF and G-CSF by two human astroglial cell lines. In the present study, we examined the effects of TNF-alpha on the production of GM-CSF and G-CSF by U87MG, a human astroglial cell line that constitutively expresses GM-CSF and G-CSF, and U373MG, a second human astroglial cell line that does not produce CSF. We demonstrate that U87MG can be induced to increase its production of GM-CSF and G-CSF by exposure to TNF-alpha while U373MG is induced to produce GM-CSF but not G-CSF. These responses, measured by accumulation of elevated levels of CSF protein and mRNA, are rapid and sensitive. The implications of these findings to the immunopathogenesis of central nervous system infections are discussed.     

Differentiation inducing effects of vesnarinone on human glioma cells

The effects of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone) on the growth of glioma cells were examined in vitro. Vesnarinone at a dose of 100 mug/ml suppressed the growth of four different glioma cell lines, U-251MG, U-373MG, U-87MG and A-172, by approximately 50%, with an elongation of the cytoplasmic process on day 5 of culture. The long-term culture of U-87MG with 10 mug/ml of vesnarinone was continued up to day 34. Although growth suppression was approximately 25% on day 5, it reached over 95% on day 34. An increase in the cyclic adenosine monophosphate content of glioma cells cultured with vesnarinone was observed by enzyme-linked immunosorbent assay (ELISA). The accumulation of glial fibrillary acidic protein was observed to occur with vesnarinone by ELISA. These findings suggest that vesnarinone suppressed the growth and induced differentiation of glioma cells in vitro.     

Parathyroid hormone-related protein is expressed by transformed and fetal human astrocytes and inhibits cell proliferation

Parathyroid hormone-related protein (PTHrP) and the PTH/PTHrP receptor are expressed in most normal tissues, including brain, where PTHrP is though to act locally in an autocrine or paracrine fashion. Previous in situ localization studies in adult rodents have documented CNS PTHrP expression in neurons but not in glial cells. However, a recent report describing immunoreactive PTHrP in human astrocytomas suggests that PTHrP expression may be a marker of dedifferentiation and/or malignant transformation in glial cells. To begin to test this hypothesis, constitutive and regulated PTHrP expression were examined in cultured fetal and transformed (U-373 MG) human astrocytes. PTHrP was expressed in untreated fetal astrocytes and U-373 MG cells, as determined by Northern analysis, immunocytochemical staining, and detection of PTHrP(1-84) protein in conditioned media. Epidermal growth factor and tumor necrosis factor, important growth factors in astrocyte development and malignant transformation, stimulated PTHrP expression in both cell types. Treatment of U-373 MG cells or fetal astrocytes with PTHrP(1-34) consistently inhibited cellular proliferation, as measured by [(3)H]-thymidine incorporation. These findings suggest that PTHrP, a peptide whose expression is induced by mitogens in both immature and transformed human astrocytes, may feedback inhibit cellular proliferation, an effect that may be of importance during malignant transformation as well as CNS development. Furthermore, when combined with previous evidence of PTHrP expression by PTH/PTHrP receptor-positive neurons, our demonstration of regulated PTHrP expression by receptor-positive astrocytes identifies PTHrP as a potential peptide mediator of cross-talk between glial cells and neurons.