Invasion Of U251 Cells Research Articles

Pro-invasive effect of irradiation on human glioblastoma cell line U87 and its possible mechanism

Objective To study the pro-invasive effect of irradiation on human glioblastoma cell line U87 and its possible mechanism.Methods Cultured U87 cells received different doses of irradiation (0,2,and 4 Gy).The change in cellular invasiveness was measured using the real-time cell analyzer system.The activities of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) in U87 ceils were measured by gelatin zymography before and after irradiation.The content and distribution of intracellular β-catenin after irradiation were determined by immunohistochemistry.The mRNA levels of Wnt/β-catenin target genes were measured by real-time quantitative PCR.Results After irradiation,the invasiveness of U87 cells increased significantly (P ＜ 0.01),which was dose-dependent within a certain dose range; the activities of MMP2 and MMP9 in U87 cells increased significantly (P =0.031 for MMP2 ; P =0.004 for MMP9) ;the content of β-catenin in U87 cells increased significantly (P ＜ 0.01),with translocation from the cell membrane and adherens junctions to the nucleus; the mRNA levels of Wnt/β-catenin-related genes (FZD7 and TCF1) increased significantly (P ＜ 0.01),and the transcription of Wnt/β-catenin target genes,especially those related to migration and invasion such as MMP2,MMP7,MMP9,and CD44,was significantly enhanced (P ＜ 0.05).Conclusions Irradiation can promote the invasion of glioblastoma U87 cells,possibly by activating the Wnt/β-catenin pathway and enhancing the transcription of migration-and invasion-related genes. Key words: Tumor cell line, glioblastoma; Irradiation; Invasiveness; Matrix metalloproteinase; Wnt/β-Catenin pathway

Expression of neonatal Nav1.5 in human brain astrocytoma and its effect on proliferation, invasion and apoptosis of astrocytoma cells.

In the present study, we designed and conducted a series of assays to determine the expression of voltage-gated sodium channel (VGSC) neonatal isoform Nav1.5 (nNav1.5) in human brain astrocytoma and its effect on the proliferation, migration, invasion and apoptosis of astrocytoma U251 cells. The results showed that nNav1.5 mRNA and protein were expressed in both human brain astrocytoma and normal brain tissues, but their expression levels in astrocytoma were significantly higher (P<0.05). In astrocytomas, nNav1.5 mRNA and protein levels were also different (P<0.05) and were correlated with pathological grades. The immunofluorescence confocal microscopy observations demonstrated that nNav1.5 protein was expressed in the nucleus, cytoplasm and membrane of the astrocytoma cells. After transfection, the small interfering RNA (siRNA) targeted to nNav1.5 significantly reduced the expression levels of SCN5A/nNav1.5 mRNA and protein by 57.2% (P<0.05) and 66.6% (P<0.05), respectively. The MTT, wound healing, Matrigel invasion and flow cytometric assays confirmed that following siRNA downregulation of the expression of the SCN5A/nNav1.5 gene, the invitro proliferation and invitro invasiveness of the U251 cells were significantly reduced (P<0.05 for both comparisons), and the apoptosis rate was significantly increased (P<0.05). These results revealed that nNav1.5 expression in human brain astrocytoma was upregulated, and its expression was positively correlated with the degree of malignancy. Additionally, reduced nNav1.5 expression significantly suppressed the proliferation and invasiveness of astrocytoma cells, indicating a new target in the molecular diagnosis and therapy of astrocytoma.

PAX6, a novel target of miR-335, inhibits cell proliferation and invasion in glioma cells.

Paired box 6 (PAX6), a highly conserved transcription factor, is important in glioma. However, the molecular mechanisms involved remain unclear. The present study demonstrated that the expression of PAX6 was significantly reduced with the malignancy of glioma and also identified PAX6 as a novel target of microRNA (miR)‑335, which was significantly upregulated in glioma. The inhibition of miR‑335 increased the protein expression of PAX6, whereas the upregulation of miR‑335 suppressed its expression in human glioma U251 and U87 cells. Furthermore, upregulation of miR-335 promoted U251 cell proliferation, colony formation and invasion, which was reversed by the overexpression of PAX6. Furthermore, the present study demonstrated that the effect of miR‑335 on U251 cell invasion was via the modulation of matrix metalloproteinase (MMP)‑2 and MMP‑9 expression by targeting PAX6. In conclusion, the present study demonstrated that PAX6, as a novel target of miR‑335, has an anti‑oncogenic function in glioma, and thus PAX6 may serve as a therapeutic target for glioma.

Ionizing radiation-inducible miR-494 promotes glioma cell invasion through EGFR stabilization by targeting p190B RhoGAP

MicroRNAs (miRNAs) play an important role in various stages of tumor progression. miR-494, which we had previously identified as a miRNA induced by ionizing radiation (IR) in the glioma cell line U-251, was observed to enhance invasion of U-251 cells by activating MMP-2. The miR-494-induced invasive potential was accompanied by, and dependent on, epidermal growth factor receptor (EGFR) upregulation and the activation of its downstream signaling constituents, Akt and ERK. The upregulation of EGFR by miR-494 involved the suppression of lysosomal protein turnover. Among the putative target proteins tested, p190B RhoGAP (p190B) was downregulated by miR-494, and its reduced expression was responsible for the increase in EGFR expression. A reporter assay using a luciferase construct containing p190B 3′-untranslated region (3′UTR) confirmed that p190B is a direct target of miR-494. Downregulation of p190B by small interfering RNA (siRNA) transfection closely mimicked the outcomes of miR-494 transfection, and showed increased EGFR expression, MMP-2 secretion, and invasion. Ectopic expression of p190B suppressed the miR-494-induced EGFR upregulation and invasion promotion, thereby suggesting that p190B depletion is critical for the invasion-promoting action of miR-494. Collectively, our results suggest a novel function for miR-494 and its potential application as a target to control invasiveness in cancer therapy.

Targeting microRNA-23a to inhibit glioma cell invasion via HOXD10

Glioma is the most frequent primary brain tumor. Recently, the upregulation of microRNA (miR)-23a was found to be associated with glioma, but the molecular mechanism by which miR-23a promotes glioma growth remains to be unveiled. In the present study, we found that miR-23a was significantly upregulated in glioma tissues compared to their matched adjacent tissues. miR-23a was also highly expressed in glioma cell lines SHG44, U251, and U87 cells. Moreover, we identified homeobox D10 (HOXD10) as a novel target for miR-23a. The expression of HOXD10 was significantly reduced in glioma tissues and cell lines, and miR-23a negatively regulates the protein expression of HOXD10 in U251 and U87 cells. We further showed that miRNA-23a promoted U251 and U87 cell invasion, at least partially, by directly targeting HOXD10 and further modulating MMP-14. These findings suggest that miR-23a may serve as a promising therapeutic target for glioma.

MicroRNA-203 inhibits the proliferation and invasion of U251 glioblastoma cells by directly targeting PLD2.

MicroRNAs (miRNAs) have been demonstrated to be important in the development and progression of various types of cancer. However, the exact roles of certain anti‑oncogenic miRNAs in human malignant gliomas remain to be elucidated. The present study aimed to reveal the expression of microRNA‑203 (miR-203) in normal brain tissues and gliomas, and to investigate the role of miR-203 in cell proliferation and migration in human glioblastoma U251 cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of miR-203 in high WHO grade glioma tissues was significantly decreased compared with low WHO grade glioma tissues and normal brain tissues, and its expression demonstrated a decreasing tendency with ascending WHO grades. The transfection of the miR-203 mimic into U251 cells markedly downregulated the expression of phospholipase D2 (PLD2), which was identified as a direct target of miR-203. Furthermore, miR-203 overexpression significantly suppressed the proliferation and invasion of U251 cells, while the overexpression of PLD2 abrogated these effects induced by the miR-203 mimic. In conclusion, the present study demonstrated the clinical significance of miR-203 in gliomas and suggested that miR-203 was able to inhibit the proliferation and invasion of glioma cells, partially at least via suppressing the protein expression of PLD2. Thus, miR-203 may be a novel candidate for the development of therapeutic strategies for gliomas.

The Effect and Mechanism of CXCR4 Silencing on Metastasis Suppression of Human Glioma U87 Cell Line

Tumor metastasis is the major cause of treatment failure and poor prognosis of glioma. Inhibiting metastasis has become an important therapeutic strategy for glioma treatment. CXCR4 has been proved to play an important role in the occurrence and development of tumors. In order to illustrate the effect of CXCR4 on glioma metastasis, we investigated the role of CXCR4 in U87 cells metastasis based on the CXCR4 silencing tumor cells. In this study, we found that CXCR4 silencing could suppress U87 cells invasion and adhesion potential, production of TGF-β1, IL-6, and IL-8, and blocked the G0/G1 phase of the cell cycle. We also found that CXCR4 silencing could up-regulate the mRNA and protein expression of p53, p21, and E-cadherin, and down-regulate the mRNA and protein expression of CD44 and MMP-2/-9. Meanwhile, CXCR4 silencing could decrease the phosphorylation of p-AKT and transcription activity of NF-κB promoter, and increased the phosphorylation of PTEN. The results provided a new research basis for the further study of CXCR4 gene, the screening of human glioma, as well as the target treatment for glioma and its prognosis.

Dihydroartemisinin suppresses cell proliferation, invasion, and angiogenesis in human glioma U87 cells

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, which is a well tolerated and effective drug for malaria treatment, has shown potent antitumor ability. This study is explored to evaluate whether DHA can inhibit glioma cell proliferation, invasion, and glioma cell mediated angiogenesis. We determined the glioma U87 cell proliferation by Cell Counting Kit-8 (CCK-8) assay, fluorescence, and flow cytometry (FCM). The invasion and migration of U87 cell were tested by wound-scratch assay and martrigel-transwell methods, while its angiogenesis tube formation assay was tested with reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Our data suggested that DHA inhibited cell viability in a dose- and time-dependent manner, triggered a stringent G1 cell cycle arrest, and induced cell apoptosis of glioma U87 cells. Tube formation assays showed that DHA significantly decreased glioma cell tube formation in human umbilical vein endothelial cell (HUVC), and suppressed vascular endothelial growth factor (VEGF) mRNA expression and its release in U87 cells. In addition, wound-scratch assay and martrigel-transwell showed that DHA decreased U87 cell invasion and migration in vitro. These results indicated that DHA might be a valuable candidate for treatment of human glioma. Key words: Dihydroartemisinin, glioma, proliferation, invasion, angiogenesis.

Overexpression of SASH1 related to the decreased invasion ability of human glioma U251 cells

The purpose of this study was to investigate the impact of SAM- and SH3-domain containing 1 (SASH1) on the biological behavior of glioma cells, including its effects on cellular growth, proliferation, apoptosis, invasion, and metastasis, and thereby to provide an experimental basis for future therapeutic treatments. A pcDNA3.1-SASH1 eukaryotic expression vector was constructed and transfected into the U251 human glioma cell line. Using the tetrazolium-based colorimetric (MTT) assay, flow cytometry analyses, transwell invasion chamber experiments, and other methods, we examined the impact of SASH1 on the biological behaviors of U251 cells, including effects on viability, cell cycle, apoptosis, and invasion. Furthermore, the effect of SASH1 on the expression of cyclin D1, caspase-3, matrix metalloproteinase (MMP)-2, MMP-9, and other proteins was observed. Compared to the empty vector and blank control groups, the pcDNA3.1-SASH1 group of U251 cells exhibited significantly reduced cell viability, proliferation, and invasion (p < 0.05), although there was no difference between the empty vector and blank control groups. The pcDNA3.1-SASH1 group demonstrated a significantly higher apoptotic index than did the empty vector and blank control groups (p < 0.05), and the percentage of apoptotic cells was similar between the empty vector and blank control groups. In addition, the pcDNA3.1-SASH1 group expressed significantly lower protein levels of cyclin D1 and MMP-2/9 compared to the control and empty vector groups (p < 0.05) and significantly higher protein levels of caspase-3 than the other two groups (p < 0.05). Cyclin D1, caspase-3, and MMP-2/9 expression was unchanged between the empty vector and blank control groups. SASH1 gene expression might be related to the inhibition of the growth, proliferation, and invasion of U251 cells and the promotion of U251 cells apoptosis.

Human U87 Astrocytoma Cell Invasion Induced by Interaction of βig-h3 with Integrin α5β1 Involves Calpain-2

It is known that βig-h3 is involved in the invasive process of many types of tumors, but its mechanism in glioma cells has not been fully clarified. Using immunofluorescent double-staining and confocal imaging analysis, and co-immunoprecipitation assays, we found that βig-h3 co-localized with integrin α5β1 in U87 cells. We sought to elucidate the function of this interaction by performing cell invasion assays and gelatin zymography experiments. We found that siRNA knockdowns of βig-h3 and calpain-2 impaired cell invasion and MMP secretion. Moreover, βig-h3, integrins and calpain-2 are known to be regulated by Ca2+, and they are also involved in tumor cell invasion. Therefore, we further investigated if calpain-2 was relevant to βig-h3-integrin α5β1 interaction to affect U87 cell invasion. Our data showed that βig-h3 co-localized with integrin α5β1 to enhance the invasion of U87 cells, and that calpain-2, is involved in this process, acting as a downstream molecule.

Lentiviral expression of anti-microRNAs targeting miR-27a inhibits proliferation and invasiveness of U87 glioma cells

Glioma is a highly fatal malignant disease and its treatment options are limited. microRNAs represent a novel target for the treatment of cancer. In the present study, we used a lentiviral vector to stably express anti-microRNAs targeting the oncogenic miR-27a in U87 glioma cells. The stable expression of anti-miR-27a significantly reduced the proliferation and increased the accumulation of U87 cells in the sub-G1 phase as determined by Cell Counting kit-8 (CCK-8) assays and flow cytometry, respectively. Results from the Matrigel Transwell assay also indicated that the inhibition of miR-27a substantially impaired the invasiveness of U87 cells. By combining bioinformatic and proteomic approaches, we identified the mRNAs of 8 proteins upregulated in anti-miR-27a-expressing U87 cells as putative direct targets of miR-27a. Collectively, these data suggest that the lentiviral expression of anti-miR-27a is a feasible approach for the suppression of malignant phenotypes of glioma cells.

Effect of silencing HIF-1α on proliferation, invasion and migration of glioblastoma U87 cells

The objective of the study was to evaluate the effect of silencing hypoxia-inducible factor 1α (HIF-1α) on proliferation, invasion, and migration of glioblastoma U87 cells. HIF-1α-shRNA lentiviral vector was designed for liposome-mediated transfection into U87 cells. The efficiency of interference was assessed by RT-PCR and Western blot. Cell proliferation was measured by MTT assay. Cell migration was observed by the migration test. The capabilities of invasion and migration were detected using the Transwell model. The research involved experiments in the interference group (shRNA transfected), the control interference group (empty vector transfected), and the untreated (non-transfected) group. Compared with the control interference group and the untreated group, the expressions of HIF-1α mRNA and protein were significantly down-regulated, and the proliferation and invasion of U87 cells were significantly inhibited in the interference group. HIF-1α mRNA and protein are effectively suppressed by HIF-1α-shRNA in U87 cells, which appears to inhibit proliferation, invasion, and migration of U87 cells.

MiR-221/222 is the regulator of Cx43 expression in human glioblastoma cells

The miR-221/222 cluster is significantly upregulated in malignant glioma cells and regulates the expression of multiple genes associated with glioma cell proliferation, invasion and apoptosis, which was shown in our previous studies. Cx43 has been identified as a tumor suppressor and major component for the establishment of gap junction intercellular communication (GJIC) in glial cells, which is frequently reduced or deleted in high-grade gliomas. According to bioinformatic analysis, connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the present study was to validate Cx43 as a target gene of miR-221/222 and to determine whether overexpression of miR-221/222 is one of the molecular mechanisms for the reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine method. Northern blot analysis was conducted to detect the expression of the miR-221/222 cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence staining. Scrape loading and dye transfer (SLDT) assays were used for examination of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell assays, respectively. Cell cycle kinetics and apoptosis were determined with flow cytometry. We found that expression of the miR-221/222 cluster was significantly reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222, and the GJIC deficiency in parental U251 cells was re-established. Moreover, the luciferase activity determined by the luciferase reporter assay was enhanced in AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed while apoptosis was induced. We conclude that miR-221/222 function as oncogenic microRNAs in human gliomas, at least in part, by targeting Cx43.

Abstract 562: Multi-Kinase Inhibitor: AFPep inhibits the growth and invasion of U87 glioblastoma cells through inhibition of FAK, MAPK, and PKC

Abstract INTRODUCTION: Glioblastomas are the most frequent tumors of the central nervous system. Patients with a diagnosis of glioblastomas have poor prognosis, and new therapeutic agents are in demand for management of this disease. Here, we report on a novel multi-protein kinase inhibitor, AFPep, with regard to its effects on the growth, invasion, and localization in U87 cells. METHODS: Proliferation/Invasion Assay: Cells were seeded into wells of plastic tissue culture plates and treated with various concentrations of AFPep. Crystal violet method was used to determine cell numbers. The Matrigel chemoinvasion assay was used to determine the effect of AFPep on U87 cell invasion. During Xenograft growth assay, U87 cells were solidified in fibrin clot and implanted under the kidney capsule of severe combined immunodeficient (SCID) mice. Various doses of AFPep were administrated i.p and tumor growth was monitored during survival laparotomy. Localization assay was performed by utilizing fluorescent-labeled AFPep. Multi-immunoblotting Assay was performed using Kinexus, the KinetworksTM methodology to screen for protein kinase expression profile in GBM before and after AFPep treatment. RESULTS: AFPep inhibited the growth of U87 cells in culture with it is maximal effect at 10−7 M. Higher concentrations of AFPep (10−6M to 10−4M) inhibited the invasion of U87 cells in a dose dependent manner. Treatment of SCID mice bearing U87 xenografts with 4 mg/Kg/day AFPep resulted in a 50% inhibition of tumor growth. Further, AFPep-Bodipy appeared to be localized in mitochondria and interferes with phosphorylation of FAK, MAPK, and c-kit. CONCLUSION: AFPep has an inhibitory effect on U87 cells. All studies to date have shown no toxicity. As such AFPep is expected to be tolerated and may be a new agent for the treatment of malignant glioblastomas Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2011-562

Wnt‐5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP‐2

Wnts are secreted ligands that consist of 19 members in humans, regulate cell proliferation, differentiation, motility and fate in many stages including the embryonic stage and tumorigenesis. Wnts bind to cell surface receptors named Frizzleds and LRPs, and transduce their signals through β-catenin-dependent and -independent intracellular pathways. Gliomas are one of the most common intracranial tumors. Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. However, the molecular mechanisms that stimulate the invasion of glioma cells are not fully understood. We established two cell lines from human glioma cases and analyzed the expression of all Wnt and Frizzled members in these cell lines and other well-known glioma cell lines by real-time PCR study. The mRNA of Wnt-5a and -7b and Frizzled-2, -6 and -7 were overexpressed in glioma cells. The elevation of Wnt-5a expression was most remarkable. Although Wnt-5a is reported to have oncogenic and antioncogenic activity in several cancers, the role of Wnt-5a signaling in human glioma cells remains unclear. Immunohistochemical study also revealed high expression of Wnt-5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt-5a expression was correlated with the clinical grade. Knockdown of Wnt-5a expression suppressed migration, invasion and expression of matrix metalloproteinase-2 of glioma cells. Reciprocally, treatment with purified Wnt-5a ligand resulted in stimulation of cell migration and invasion. MMP-2 inhibitor suppressed the Wnt-5a-dependent invasion of U251 cells. These results suggested that Wnt-5a is not only a prognostic factor but also a therapeutic target molecule in gliomas for preventing tumor cell infiltration.

Resveratrol represses YKL-40 expression in human glioma U87 cells

BackgroundGlioblastoma multiforme (GBM) is the most malignant intracranial tumour that develops in both adults and children. Microarray gene analyses have confirmed that the human YKL-40 gene is one of the most over-expressed genes in these tumours but not in normal brain tissue. Clinical studies have shown that serum YKL-40 levels are positively correlated with tumour burden in addition to being an independent prognostic factor of a short relapse-free interval as well as short overall survival in patients with various cancers. Our previous study revealed that YKL-40 was closely correlated with the pathological grades of human primary astrocytomas and played a crucial role in glioma cell proliferation. Hence, YKL-40 could be an attractive target in the design of anti-cancer therapies.MethodsCell viability and invasion assays were performed to detect the cell proliferation and invasive ability of U87 cells induced by resveratrol (3, 5, 4'-trihydroxystilbene; Res) or YKL-40 small-interfering RNAs (siRNAs). In addition, the luciferase assay, real-time RT-PCR, western blotting, and ELISA were used to measure YKL-40 promoter activity, mRNA, and protein expression, respectively. The expressions of phosphor-ERK1/2 and ERK1/2 were determined by western blotting.ResultsRes inhibited U87 cell proliferation and invasion in vitro and repressed YKL-40 in U87 cells by decreasing the activity of its promoter and reducing mRNA transcription and protein expression in vitro. YKL-40 siRNA treatment also impaired the invasiveness of U87 cells. When U87 cells were cultured with 20 μM PD98059 (an ERK1/2 inhibitor) alone, with 20 μM PD98059 and 100 μM Res, or with 100 μM Res alone for 48 h, YKL-40 protein expression decreased most significantly in the Res-treated group. PD98059 partially reversed the decrease of YKL-40 protein expression induced by Res. Furthermore, phosphor-ERK1/2 expression was reduced by Res treatment in a time-dependent manner.ConclusionsWe demonstrated for the first time that Res represses YKL-40 expression in vitro; in addition, the ERK1/2 pathway is involved in this repression. This finding could extend the prospective use of Res in glioma research and enlarge the armamentarium for treating gliomas.

Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells

Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose-dependent manner. BrdU and tube formation assays indicated that gallic acid significantly decreased glioma cell proliferation and tube formation in mouse brain endothelial cells, respectively. In addition, gallic acid decreased U87 cell invasion in vitro. Western blot analysis showed that expression of ADAM17, p-Akt and p-Erk was suppressed by gallic acid in both U87 and U251n cell lines. These data suggest that suppression of ADAM17 and downregulation of PI3K/Akt and Ras/MAPK signaling pathways may contribute to gallic acid-induced decrease of invasiveness. Gallic acid may be a valuable candidate for treatment of brain tumor.

Expression of matrix metalloproteinase-26 promotes human glioma U251 cell invasion in vitro and in vivo

MMP-26 is a novel member of the MMP family and is widely expressed in cancer cells of epithelial origin. Published research shows that MMP-26 contributes to tumor development and to the restoration of tissue injury. In this study, in order to identify the functions of MMP-26 that contribute to the biological phenotype and behavior of non-epithelial human glioma U251 cells, we established an MMP-26 overexpressing tumor cell model using gene transfection. We then used these cells to investigate the role of MMP-26 in tumor progression. Adherence and spreading assay, wound healing assay, Boyden chamber invasion assay, and in vivo tumorigenicity assay were performed to analyze the invasion ability of MMP-26 transfected U251 cells. Microvessel density analysis and tumor cell induced angiogenesis assay were employed to detect the function of MMP-26 in angiogenesis. Results showed that the spreading cell ratio of MMP-26 transfected cells was significantly higher than parental U251 cells. The relative migration distance of MMP-26 transfected cells on Matrigel was significantly higher than that of parental U251 cells. The Boyden chamber assay showed that MMP-26 could significantly enhance the ability of U251 cells to invade through Matrigel. MMP-26 could also enhance the local invasion ability of U251 cells in vivo. There was a significant increase of the microvessel density of tumor tissue derived from MMP-26 transfected U251 cells. The vessel numbe induced by MMP-26 transfected U251 cells in nude mice was also significantly higher than that induced by parental U251 cells. In conclusion, we successfully established an MMP-26 overexpressing cell model and confirmed that MMP-26 contributed to U251 cell invasion and migration in vitro. We also demonstrated that MMP-26 plays an important role in local invasion, and angiogenesis.

The phosphorylation of ephrin‐B2 ligand promotes glioma cell migration and invasion

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan-Meier analysis demonstrated ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin-B2 were high in GBM. Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin-B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin-B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a strong predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.

Glioblastoma and endothelial cells cross-talk, mediated by SDF-1, enhances tumour invasion and endothelial proliferation by increasing expression of cathepsins B, S, and MMP-9

Malignant glioma is characterized by rapid proliferation, high invasiveness into the surrounding brain and increased vascularity. The aim of the study was to explain the observation that glioblastoma invasion often occurs along existing vasculature, suggesting interactions between the two types of cells. Using the in vitro model, we demonstrate that co-culturing of U87 (human glioblastoma) cells with HMEC-1 (human microvascular endothelial) cells increases the invasiveness of the U87 cells. The enhanced invasiveness correlates with increased expression of MMP-9 in both U87 and HMEC-1 cells, increased expression of cysteine cathepsins B and S and down-regulation of endogenous cell adhesion molecule NCAM in U87 cells. On the other hand, U87 tumour cells significantly enhance the proliferation of co-cultured endothelial cells by a mechanism involving cathepsin B, but not cathepsin S. Furthermore, we demonstrated that increased cell expression and activity of MMP-9 in cell microenvironment is mediated via secretion of SDF-1 by HMEC-1 cells. Selective SDF-1 inhibition impaired the enhanced U87 cell invasion, mostly via down-regulation of MMP-9, but did not alter cathepsin B, although the latter is more relevant for the invasion of U87 cells in mono-culture. Taken together, our study suggests that glioblastoma cells may be attracted by endothelial cells, enhancing their proliferation and underlines the importance of SDF-1, cathepsin B and MMP-9 in the cross-talk between these cells in normoxic conditions. This notion contributes to better understanding and suggests further investigations of the paracrine mechanisms, regulating glioma angiogenesis.