CD44 expression and hyaluronic acid binding of malignant glioma cells

The mechanisms leading to rapid invasive growth of malignant gliomas are poorly understood. Expression of the hyaluronic acid (HA) receptor CD44 and adhesion to HA are involved in invasive properties. Our previous studies have shown that malignant glioma cells are able to adhere to extracellular HA. Here we investigated expression of the hyaluronic acid receptor CD44 protein in five human (T98G, A172, U87MG, 86HG39, 85HG66) and two rat (C6, 9L) glioma cell lines. Influence of anti-CD44 antibody and hyaluronidase-preincubation on the HA-binding was determined using HA/BSA (bovine serum albumin)-coated culture plates. While all gliomas were highly positive for CD44 with no differences in the number of positive staining cells, median fluorescence intensity decreased as follows: C6>T98G>9L>85HG66> 86HG39>A172> U87MG. Using HA/BSA coated culture plates the relative levels of specific adhesion to HA were determined as T98G>A172>9L>86HG39>U87MG> 85HG66. C6 cells failed to bind HA specifically. Incubation with anti-human-CD44 MAb significantly decreased HA-adhesion of T98G, A172, 85HG66 and U87MG human glioma cells. However the binding capacity was completely blocked only in 85HG66 cells. The three other cell lines kept a specific HA-adhesion after saturation of the receptor. Hyaluronidase pretreatment markedly enhanced HA-adhesion of C6 and 9L rat glioma cells. These results suggest that (i) HA-adhesion of malignant glioma cells is mainly, but not only, mediated by CD44, (ii) expression of CD44 does not correspond with adhesion capacity and (iii) cell-bound glycosaminoglycans may influence glioma cell adhesion to extracellular HA.     

Role of fibronectin-stimulated tumor cell migration in glioma invasion in vivo: clinical significance of fibronectin and fibronectin receptor expressed in human glioma tissues

In order to clarify the role of fibronectin in glioma invasion in vivo, we analyzed the relationship between fibronectin-stimulated cell migration and adhesion in 14 primary glioma cells and the expression of fibronectin and the fibronectin receptor in the corresponding tumor tissues. The tumors comprised nine glioblastomas (GB) and five anaplastic gliomas (AG) consisting of two astrocytomas, two oligoastrocytomas and one ependymoma. All glioma cells tested in the primary cell culture were found to migrate to fibronectin in a dose-dependent manner. The extent of cell migration to fibronectin was not significantly different for the GB and AG groups. On the other hand, cell adhesion to fibronectin in the AG was much stronger than that in the GB group. Immunohistochemistry demonstrated that fibronectin positively stained in the extra-cellular matrix (ECM) in eight cases and that the fibronectin receptor was positive in tumor cell membranes in 10 cases. In addition, cellular fibronectin isoforms containing ED-A and ED-B sequences were found to be immunolocalized in the tumor cells and the ECM of GB. These isoforms were also specifically expressed in tumor vessels within tumor tissues, but not in those within normal brain tissues. Cell migration tended to be expressed more strongly by glioma cells derived from tumor tissues in which fibronectin was posi-tively immunolocalized in the ECM than from tissues with negative fibronectin in the ECM. Four glioma cells derived from GB whose tumor cells did not positively stain for fibronectin receptors migrated much less extensively to fibronectin than other glioma cells whose tissues showed positive staining for the fibronectin receptor. Of these four GB, two had loss of heterozygosity in the locus of fibronectin receptor b1 gene. These results suggest that fibronectin deposited in the extracellular matrix of tumors, which can be derived from both plasma and the tumor cell itself, strongly promotes the migration of glioma cells, and that expression of the fibronectin receptor may play a critical role in the biological behavior of the tumor cells, particularly in fibronectin-stimulated cell migration in vivo.© Kluwer Academic Publishers 1998     

血管细胞黏附分子-1增强人脑胶质瘤细胞系迁移和侵袭能力

目的探讨血管细胞黏附分子-1(VCAM-1)在脑胶质瘤细胞迁移侵袭能力中作用。方法用慢病毒p SGU6/GFP/Neo介导VCAM-1的shRNA、慢病毒EF1a-GFP/puro介导VCAM-1过表达载体、划痕迁移、Transwell侵袭、蛋白免疫印迹(Western blot)和细胞染色等实验技术和方法,观察了VCAM-1蛋白表达水平对人脑胶质瘤T98G和U251细胞系细胞迁移和侵袭能力的影响。其中,T98G细胞分为空白对照组、空载体对照组、乱序对照组和实验组(抑制VCAM-1蛋白表达水平组),U251细胞分为空白对照组、空载体对照组和实验组(过表达VCAM-1组),每组6个复孔。结果首先利用慢病毒介导VCAM-1的shRNA和过表达载体建立了稳定低表达VCAM-1的T98G细胞和稳定过表达VCAM-1的U251细胞。稳定低表达VCAM-1的T98G细胞划痕恢复能力(迁移能力)明显减弱(P0.01);而稳定过表达VCAM-1的U251细胞迁移能力明显提高(P0.05)。同样,稳定低表达VCAM-1的T98G细胞侵袭能力显著减弱(P0.05);而稳定过表达VCAM-1的U251细胞侵袭能力明显增强(P0.01)。结论VCAM-1可显著增强人脑胶质瘤细胞系细胞的迁移和侵袭能力。     

乙酰辅酶A羧化酶1对胶质瘤细胞系U87细胞增殖、迁移和侵袭的影响

目的 探讨乙酰辅酶A羧化酶1（ACC1）对人胶质瘤细胞系U87细胞增殖、迁移及侵袭的作用。 方法 Western blotting检测人胶质瘤细胞系U87、U251及U373中ACC1的表达;构建ACC1过表达质粒载体,将过表达ACC1质粒载体瞬时转染至U87细胞中;Western blotting检测转染后U87细胞中ACC1表达情况;MTT实验检测过表达ACC1对U87细胞增殖的影响;Transwell迁移和侵袭实验分别检测过表达ACC1对U87细胞迁移和侵袭的影响;划痕实验检测过表达ACC1对U87细胞划痕愈合能力的影响;Western blotting检测相关蛋白表达变化。 结果 与人胶质瘤细胞系U251和U373相比,U87细胞中ACC1表达较低;ACC1过表达抑制U87细胞增殖（P<0.01）;ACC1过表达抑制U87细胞迁移、侵袭和划痕愈合能力（P<0.01）;ACC1过表达迁移和侵袭相关蛋白波形蛋白(vimentin)、纤维连接蛋白(fibronectin)和尿激酶型纤溶酶原激活剂(uPA)表达下调（P<0.01）,凋亡抑制蛋白Bcl-2和细胞周期蛋白(cyclin) B、cyclin D表达下调（P<0.01）,p-STAT3蛋白表达下调（P<0.01）,细胞周期蛋白P21表达上调（P<0.01）。 结论 过表达ACC1可能通过抑制STAT3活性,抑制人胶质瘤细胞的增殖、迁移和侵袭。     

整合素β1及纤连蛋白、层粘连蛋白对胶质瘤细胞浸润性的影响

目的 探讨整合素β1和纤连蛋白（FN）、层粘连蛋白（LN）对人胶质瘤浸润性的影响和作用机制。方法 以U251人胶质母细胞瘤（U251MG）细胞为研究对象,通过细胞黏附实验、迁移实验和体外侵袭实验,检测整合素β1及LN、FN对人恶性胶质瘤细胞黏附、迁移和转移能力的影响。通过荧光染色结合激光扫描共聚焦显微镜观察和扫描电镜方法,观察细胞微丝数量、分布和细胞表面伪足情况,比较整合素β1及FN、LN对微丝骨架的影响。结果 （1）FN对U251MG细胞黏附能力无明显影响,但抗整合素β1抗体可减少U251MG细胞的黏附数量（P〈0．01）;LN增加U251MG细胞黏附能力（P〈0．01）,抗整合素β1抗体对此作用影响较小。（2）抗整合素β1抗体减弱U251MG细胞在FN的运动、迁移能力（P〈0．05）。（3）U251MG细胞内可见清晰的微丝结构,FN、LN使细胞内纤维型肌动蛋白（F-actin）形成束状纤维,粗壮而密集;抗整合素B1抗体处理的细胞内,难以见到清晰的细胞微丝骨架,并常见大量絮团状的F-actin。（4）扫描电镜观察显示,FN、LN使细胞表面的伪足数量明显增加,而抗整合素β1抗体使细胞伪足数量明显减少,甚至消失。（5）FN和抗整合素β1抗体对U251MG细胞的体外侵袭能力无明显影响;LN可促进U251MG细胞的体外侵袭能力,抗整合素β1抗体可抑制这种作用（P〈0．01）。结论 （1）U251MG细胞通过整合素β1和FN相互作用,改变细胞微丝骨架、伪足结构和数量而促进U251MG细胞的运动、迁移能力。（2）整合素β1参与了LN介导的U251MG细胞体外侵袭作用。     

Fibronectin expression correlates with U937 cell adhesion to migrating bovine aortic endothelial cells in vitro.

A blood vessel's response to denudation injury will determine its final luminal diameter as well as its function. The synthesis, deposition, and remodeling of extracellular matrix components and migration by vascular endothelial cells are major factors in determining luminal diameter, cellular proliferative and migratory responses, and mononuclear cell adhesion at sites of injury. Previously, we have shown that after in vivo and in vitro denudation injury, endothelial cell migration is dramatically influenced by the amount of fibronectin synthesized and deposited by the responding endothelial cell population. The aim of this study was to elucidate the roles of fibronectin in modulating mononuclear cell adhesion to the endothelial cell population during in vitro migration. In this report we demonstrate that U937 cell binding to the migrating fronts of endothelial cell monolayers is modulated by the amount of fibronectin synthesized and deposited by the endothelial cells. Agents which increase fibronectin deposition, such as transforming growth factor-beta 1, elicit greater U937 cell adhesion. Manipulations that decrease fibronectin deposition, such as transfection and overexpression of pp60c-src proto-oncogene in endothelial cells, reduce U937 cell adhesion. These results suggest that changes in endothelial cell extracellular matrix synthesis and deposition modulate, in part, the adhesive properties of the vessel wall after injury. In turn, the intensity and duration of mononuclear cell adhesion at sites of vessel wall injury determines, in part, the vessel wall response.     

Podoplanin increases migration and angiogenesis in malignant glioma

Expression of podoplanin in glial brain tumors is grade dependent. While serving as a marker for tumor progression and modulating invasion in various neoplasms, little is known about podoplanin function in gliomas. Therefore we stably transfected two human glioma cell lines (U373MG and U87MG) with expression plasmids encoding podoplanin. The efficacy of transfection was confirmed by FACS analysis, PCR and immunocytochemistry. Cells were then sorted for highly podoplanin expressing cells (U373P^high/U87P^high). Transfection did not influence the production of pro-angiogenic factors including VEGF, VEGF-C and D. Also, expression of VEGF receptors (VEGFR) remained unchanged except for U87P^high, where a VEGFR3 expression was induced. U373P^high showed significantly reduced proliferation as compared to mock transfected group. By contrast, podoplanin significantly increased migration and invasion into collagen matrix. Furthermore, conditioned media from P^high glioma cells strongly induced tube formation on matrigel. In conclusion, podoplanin increased migration of tumor cells and enhanced tube formation activity in endothelial cells independent from VEGF. Thus, podoplanin expression may be an important step in tumor progression.     

RETRACTED: Alpinumisoflavone suppresses human Glioblastoma cell growth and induces cell cycle arrest through activating peroxisome proliferator-activated receptor-γ

As a common subtype of malignant gliomas, glioblastoma multiforme (GBM) is associated with poor prognosis. This study is aimed to examine the anticancer activities of alpinumisoflavone (AIF) and its underlying mechanisms. Our results demonstrated that AIF inhibited the proliferation of GBM cells (U373 and T98G) in a time and dose-dependent manner. In addition, flow cytometry analysis not only confirmed AIF arrested cell cycle at the G0/G1 phase but also the induced apoptosis of U373 and T98G cells. Western blotting also confirmed that AIF altered the expression levels of cell cycle-related proteins. Further mechanism studies revealed that AIF inhibited cell proliferation, induced G0/G1 phase arrest and induced apoptosis of U373 and T98G cells through activating PPARγ, as evidenced by the fact that GW9662 (PPARγ inhibitor) could effectively reverse the effects of AIF on U373 and T98G cells. Furthermore, the in vivo study also revealed that AIF suppressed tumor growth and caused cell cycle arrest. Collectively, these results highlighted the potential use of AIF in the treatment of GBM.     

CBX8过表达或沉默对人神经胶质瘤细胞增殖和凋亡的影响

目的:探究染色质结构域蛋白8(chromodomain protein 8,CBX8)对人神经胶质瘤细胞增殖与凋亡的作用。方法:Western blot和RT-qPCR检测组织及细胞系中CBX8的表达。构建过表达CBX8和沉默CBX8载体,转染神经胶质瘤细胞T98G和U87MG,分别用MTT法和BrdU实验检测细胞增殖,流式细胞术检测细胞凋亡。结果:与正常脑组织和星形胶质细胞相比,神经胶质瘤组织及细胞中的CBX8蛋白和mRNA水平明显上升。在T98G和U87MG细胞中,过表达CBX8均促进细胞增殖,抑制细胞凋亡,并上调Rb/E2F1的表达水平,而沉默CBX8则作用相反。sh-E2F1转染细胞之后,cyclin D1的表达以及Bcl-2/Bax的比值降低。结论:CBX8可能通过Rb/E2F1通路调节胶质瘤细胞的增殖和凋亡。     

SEPT7基因抑制人胶质瘤细胞系U251MG侵袭的研究

目的 研究SEPT7基因对人胶质瘤细胞系U251MG侵袭的抑制作用及其可能的分子机制.方法 以腺病毒为载体转导SEPT7(rAd5-SEFF7)入U251人脑胶质瘤细胞系;Transwell法和3-D Matrigel法观察U251胶质瘤细胞侵袭能力的变化,划痕实验和2-D Matrigel法观察细胞迁移能力的变化.应用蛋白印记检测MMP2,MMP9,MT1-MMP,TIMP1和TIMP2的表达变化,蛋白印记和免疫荧光检测整合素αvβ3的表达,以及应用激光扫描共聚焦显微镜观察细胞骨架蛋白tubulin-α结构的变化.结果 转染SEPT7后U251MG细胞的侵袭和迁移能力明显受到抑制、细胞MMP2、MMP9、MT1-MMP和整合素αvβ3的表达下调、TIMP1和TIMP2的表达则上调;肿瘤细胞的微管蛋白tubulin-α结构出现了重新分布,发生了扭曲及聚集现象,接近于正常的非肿瘤细胞的tubulin-α结构.结论 SEPT7基因可以抑制胶质瘤细胞的侵袭和迁移能力,其分子机制可能通过逆转MMPs/TIMPs的失衡状态,降低整合素αvβ3的表达,以及改变细胞骨架tubulin-α的结构而实现的.SEPT7可作为基因治疗胶质瘤的重要候选基因.     

ATAD2对人胶质瘤细胞系转移及上皮间质转换的影响

目的探讨三磷酸腺苷酶家族蛋白2(ATAD2)对人胶质瘤细胞的影响。方法用real-time PCR及Western blot检测ATAD2在人胶质瘤细胞系U87MG、U251和正常人星形胶质细胞的表达。U87MG和U251分为对照组(control)、脂质体对照组(mock)、ATAD2 siRNA(转染特异性siRNA)和ATAD2 overexpression(过表达ATAD2)4组,MTT法、Transwell实验观察细胞增殖和侵袭迁移。Western blot检测上皮间质转换标志蛋白上皮钙黏素、神经钙黏素和波形蛋白的表达。结果 U87MG和U251细胞ATAD2高表达。与对照相比,ATAD2 siRNA抑制U87MG和U251细胞增殖、侵袭、迁移、神经钙黏素及波形蛋白表达,促进上皮钙黏素表达(P0.05);ATAD2 overexpression促进细胞增殖、侵袭、迁移、神经钙黏素和波形蛋白表达,降低上皮钙黏素表达(P0.05)。结论 ATAD2可能通过调节上皮间充质转换进程参与人胶质瘤细胞转移。     

miR-107抑制胶质瘤细胞的体外增殖、迁移和侵袭

目的探讨miR-107对胶质瘤细胞增殖、迁移和侵袭的调控机制。方法运用RT-qPCR检测人正常的星形胶质细胞系NHA、神经胶质瘤细胞系U87、A172、U251中miR-107和FOXK1的表达;将细胞分为miR-NC组(转染miR-NC)、miR-107组(转染miR-107 mimics)、si-NC组(转染si-NC)、si-FOXK1组(转染si-FOXK1)、miR-107+pcDNA3.1组(共转染miR-107 mimics和pcDNA3.1)和miR-107+pcDNA3.1-FOXK1组(共转染miR-107 mimics和pcDNA3.1-FOXK1);用脂质体法分别转染至U87细胞;CCK-8法检测细胞的增殖;Transwell小室实验检测细胞的迁移和侵袭;Western blot检测细胞中FOXK1的蛋白表达;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果与正常的星形胶质细胞NHA相比,神经胶质瘤细胞U87、A172、U251中miR-107表达明显下调,FOXK1表达明显上调(P<0.05);过表达miR-107、敲减FOXK1均可抑制U87细胞的增殖、迁移和侵袭;miR-107可抑制野生型FOXK1的细胞荧光活性,并负向调控FOXK1的表达;过表达FOXK1可逆转miR-107对U87细胞增殖迁移侵袭的抑制作用。结论 miR-107抑制胶质瘤细胞增殖、迁移和侵袭的作用机制可能与靶向负调控FOXK1有关,将可为胶质瘤的诊断和治疗提供靶向治疗的依据。     

Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation,migration and invasion of malignant glioma cells

Tissue factor (TF) is upregulated in several malignant diseases, including gliomas. Here, we demonstrate pronounced differences in the expression of TF and its interactors factor VII and protease-activated receptor 2 (PAR-2) in nine human glioma cell lines (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304, Hs 683) as detected by RT-PCR and Western blot analysis. Inhibition of TF signaling by a neutralizing monoclonal antibody (mAb TF9-10H10) led to significantly reduced proliferation in high-grade astroglial (MZ-18 and MZ-304) and oligodendroglial (Hs 683) cell lines abundantly expressing TF, but not in U373 cells expressing low amounts of TF. Scratch migration assays and Boyden chamber assays indicated that mAb TF9-10H10 and lentiviral knockdown of TF significantly reduced cell migration and invasion of MZ-18, MZ-304 and Hs 683 cells, both under normoxic and hypoxic conditions. Of note, all three cell lines displayed increased cell migration and invasion under hypoxic conditions (1% O₂), which was associated with enhanced expression of TF and increased phosphorylation of p44/42 mitogen-activated protein kinase (ERK1/2). Silencing of TF blocked activation of the ERK pathway, induction of TF expression and the potentiating effect of hypoxia on cell migration and invasion. RNA interference against PAR-2 abrogated the autocrine effects of TF on cell proliferation, migration and invasion, indicating that TF signals via PAR-2 in glioma cells. Our results suggest an important role for the TF/FVIIa/PAR-2/ERK axis in tumor growth and invasion of glioma and suggest that TF may be a suitable target for the development of novel therapies against high-grade glioma.     

Knockdown of TMEM45A inhibits the proliferation,migration and invasion of glioma cells

Gliomas are the most common and aggressive type of primary adult brain tumor. Although high expression and prognostic value of TMEM45A has been recently reported in various types of human tumors, the association of TMEM45A expression and glioma is still unknown. Here, we reported that TMEM45A was significantly overexpressed in glioma tissues compared to non-tumorous brain tissues. Furthermore, TMEM45A mRNA levels were gradually increased with the increasing severity of histological grade of glioma. Moreover, high TMEM45A expression level was correlated with short survival time of glioma patients. Down-regulation of TMEM45A in two glioma cell lines, U251 and U373 by transected with TMEM45A siRNA resulted in a significant reduction of cell proliferation and G1-phase arrest. Additionally, we found that suppressing of TMEM45A expression in glioma cells remarkably suppressed cell migration and cell invasion. More importantly, TMEM45A siRNA treatment significantly down-regulated the proteins promoting cell cycles transition (Cyclin D1, CDK4 and PCNA) and cell invasion (MMP-2 and MMP-9), which indicted a possible mechanism underlying its functions on glioma. In summary, our study suggests that TMEM45A may work as an oncogene and a new effective therapeutic target for glioma treatment.     

Gastrin significantly modifies the migratory abilities of experimental glioma cells

Malignant astrocytic tumors are characterized by the pronounced and diffuse migration of tumor astrocytes into the brain parenchyma. The present study shows that gastrin is a brain neuropeptide that is able to significantly modulate astrocytic tumor migration at both invasion and motility levels. In the matter of invasion, gastrin severely reduces the in vitro invasive abilities of C6 rat glioma, 9L rat gliosarcoma, and U373 human glioma cells in a collagen matrix. In vitro, gastrin also markedly modifies the motility features in both C6 and U373 cells, at least partly through a decrease in the expression of the RhoA small GTPase, and so brings about some dramatic modifications to the organization in the actin cytoskeleton. The in vitro preincubation of C6 tumor cells with gastrin significantly increases the life spans of rats stereotactically implanted with these cells as compared with the survival periods of rats implanted with gastrin-untreated C6 cells. As suggested by our in vitro experiments, these effects, observed in vivo cannot relate to only the gastrin-induced decrease in tumor astrocyte migratory abilities. Indeed, gastrin also induces immunomodulatory effects, because we observed a marked gastrin-induced recruitment of lymphocytes into C6 gliomas and 9L gliosarcomas. These data all suggest that gastrin can act as an endogenous modulator of glioma progression.     

Participation of tumor suppressors long non-coding RNA MEG3, microRNA-377 and PTEN in glioma cell invasion and migration

PurposeGlioma is a common and fatal intracranial tumor. Both miR-377 and lncRNA MEG3 are tumor suppressors. This study was performed to investigate the association between miR-377 and lncRNA MEG3 in glioma cells.MethodsU118 and U251 cell lines were incubated in Dulbecco's modified Eagle's medium supplemented with miR-377 mimics, MEG3 siRNA (si-MEG3) and/or MEG3 overexpression plasmids (pc-MEG3) for 48 h. Cell migration, invasion, apoptosis, cell cycle distribution and the expression of E26 tansformation-specific-1 (ETS-1), phosphatase and tensin homologue (PTEN), E-cadherin, N-cadherin and β-catenin were detected.ResultsMiR-377 mimics increased MEG3 expression and decreased the number of migrated and invaded U118 and U251 cells, without influence on apoptosis in both cell lines. Si-MEG3 transfection increased U118 cell migration and invasion and rescued miR-377 mimics-induced inhibitory in cell migration and invasion. Si-MEG3 decreased U118 cell apoptosis and induced G0/G1 cell cycle arrest, and pc-MEG3 increased U251 cell apoptosis via arresting cell cycle at G2/M phage. MiR-377 mimics and si-MEG3 increased the relative expression level of N-cadherin mRNA, and both si-MEG3 and pc-MEG3 increased E-cadherin in glioma cells. MiR-377 mimics increased ETS-1 mRNA in U118 cells, but decreased it in U251 cells. PTEN was increased by miR-377 mimics and si-MEG3 and decreased by pc-MEG3 in glioma cells.ConclusionsThese results suggested the link interaction of MEG3 with miR-377 and PTEN, but not functioning as the competing endogenous RNA. MiR-377 mimics and MEG3 were tumor suppressors in glioma cells through regulating PTEN expression.     

Expression of 72 kDa type IV collagenase and invasion activity of human glioma cells

Metalloproteinases, inhibitors of metalloproteinases, plasminogen activators, inhibitors of plasminogen activators and cathepsins are thought to be involved in invasion by tumor cells. Glioblastoma multiforme is highly malignant and extremely refractory to therapy. One reason is because of its highly invasive nature within the nervous system. However, it remains unclear how invasion/dissemination of glioblastoma multiforme proceeds. In this study, we attempted to determine which proteinases were responsible for the invasion activity of human glioma cell linesin vitro. Nine human glioma cell lines (NHG1, NHG2, IN157, IN301, IN500, U251, U343, T98G and CCF-STTG1) derived from patients with glioma were grown in culture and used. We compared the invasion activity of glioma cell lines in a Matrigel invasion assay system, and formulated the activity as invasion index (%). Among the nine cell lines, IN157, IN500 and U343 showed less than 10% invasion activity (low group); NHGI, IN301 and CCF-STTG1 showed 10–25% activity (intermediate group); NHG2, U251 and T98G showed more than 30% activity (high group). Addition of an inhibitor of metalloproteinases, TIMP-1, to the assay system was found to significantly inhibit invasion activity of T98G cellsP<0.01). Northern blot analysis demonstrated expression of urokinase-type plasminogen activator (uPA), tissue-type PA (tPA) and PA inhibitor-1 (PAI-1) in some of the above cell lines. Cellular levels of PAs and their inhibitor mRNA, however, appeared not to be correlated with invasion activity in most glioma cell lines except for CCF-STTG1. Expression of 72 kDa type IV collagenase (MMP-2) was much lower in IN157, IN500 and U343 than other cell lines, whereas expression of TIMP-1 was much higher in IN500 than in other cell lines. Zymographic activity was found to be comparable to MMP-2 mRNA levels in all cell lines except for CCF-STTG1. Type IV collagenolytic activity was also comparable to invasion activity in nine cell lines. These observations suggest the role of type IV collagenase and its inhibitors in determining capacity for invasion by human gliomas. However, a comprehensive analysis bothin vitro andin vivo is required to confirm the role for this enzyme in glioma cell invasiveness.     

Role of extracellular matrix proteins in regulation of human glioma cell invasion in vitro

Primary brain tumors lack the metastatic behavior that is in part believed to be promoted by the extracellular matrix (ECM) components of the basement membrane. This study was intended to examine the influence of the ECM components present in the basement membrane that may act as natural barriers to tumor cell invasion. We examined the effect of type I and type IV collagens, fibronectin, laminin, and hyaluronic acid on the migration and invasion of four established glioblastoma cell lines, SNB19, U251, UWRI, and UWR2. Lower concentrations of all the ECM components induced the migration and invasion of all the cell lines. However, in the case of SNB19, laminin inhibited both migration and invasion in a concentration-dependent manner. We have also examined the influence of individual ECM components on the migration of cells from a spheroid to a monolayer on ECM component-coated coverslips. Consistent with the invasion studies using the modified Boyden chamber assays, lower concentrations of ECM components induced the migration of cells from spheroids to monolayer. Again, laminin inhibited the migration of cells from SNB19 spheroids. These results indicate that ECM components induce the invasion of glioma cells, apart from components like laminin, which may act as natural inhibitors.     

Localization of CD44 at the Invasive Margin of Glioblastomas by Immunoelectron Microscopy

Glioblastoma multiforme is a highly invasive primary brain tumor, which is known to strongly express the CD44 cell adhesion receptor. A number of experimental studies suggest that the interaction of this receptor with extracellular matrix (ECM) proteins such as hyaluronic acid may in part mediate human glioma cell adhesion and invasion of brain tissue. Although the expression of CD44 and its spliced variants in brain tumors have been extensively studied, there have been no reports localizing its expression to the invasive margin of the tumor. The authors used immunoelectron microscopy to investigate the expression patterns of CD44 in an in vitro organotypic invasion assay. Tumor spheroids initiated from the U373 MG human glioblastoma line were confronted with fetal rat brain aggregates in a spheroid coculture system. The CD44 expression appeared at the interface between glioblastoma tumor spheroids and brain tissue, as well as in the spheroid itself. CD44 immunoreactivity was not detectable in mature 21-day fetal brain aggregates. The findings provide direct evidence that CD44 is expressed at the confrontational invasive border between glioblastomas and brain tissue, further supporting its role in glioma cell-ECM recognition and attachment.