U87 Migration Research Articles

Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti-invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia-mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF-1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia-induced HIF-1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF-1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP-2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia-induced glioblastoma cell migration and invasion via inhibition of HIF-1α. Considering the fact that overexpression of the HIF-1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.

Abstract A48: Osteopontin increases heme oxygenase-1 expression and subsequently induces cell migration in glioma cells.

Abstract Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Gliomas are associated high morbidity and mortality because these tumors are highly invasive into surrounding brain tissue, making complete surgical resection impossible. Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor in a variety of neuronal cell populations. However, the molecular mechanisms and pathologic roles underlying GDNF-induced glioma migration remain unclear. In this study, we found that application of recombinant human GDNF enhances the migration of U87 and U251 cells but not C6 cells. In addition, we found that the expression of matrix metalloproteinase-13 (MMP-13) mRNA, protein and secretion increase in response to GDNF stimulation. The GDNF-induced increase in cell migration was antagonized by MMP-13 neutralizing antibody or silencing MMP-13. We then examined the involvement of mitogen-activated protein kinases (MAPKs) in glioma cell migration induced by GDNF. GDNF-induced MMP-13 expression and glioma migration were attenuated by MEK/ extracellular signal-regulating kinase (ERK) and c-Jun N-terminal protein kinase (JNK) inhibitors, as well as ERK and JNK dominant-negative mutants. Treatment with GDNF induced MEK/ERK and JNK/c-Jun activation and increased AP-1 DNA binding activity in a time-dependent manner. Treatment with AP-1 inhibitors (tanshinone IIA and curcumin) also reduced GDNF-induced glioma cell migration. In migration-prone sublines, cells with greater migration ability had higher GDNF expression. These results indicate that GDNF enhances migration of glioma cells through the increase of MMP-13 production and is mainly regulated by the MEK/ERK and JNK, c-Jun and AP-1 pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A48.

EFFECT OF SIMVASTATIN ON GLIOMA CELL PROLIFERATION, MIGRATION, AND APOPTOSIS

In this study, we investigated the effects of simvastatin on proliferation, migration, and apoptosis in human U251 and U87 glioma cells and the underlying molecular mechanism. We used colony formation assay to test the cell proliferation, in vitro scratch assay to examine the cell migration, and caspase-3 activity assay, annexin V staining, and cytochrome C release to evaluate the cell apoptosis. Lipid raft fractions were isolated from glioma cells. Total cholesterol content assay was used to test the change of cholesterol level in lipid raft fractions. Immunocytochemistry staining was performed to detect the changes of lipid rafts in cell membranes. Western blotting analysis was performed to examine the signal transduction both in cells and in lipid raft fractions. Simvastatin inhibited proliferation and migration of U251 and U87 cells dose dependently. Simvastatin induced an increase of caspase-3 activity and annexin V staining, and down-regulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Simvastatin also decreased cholesterol content in lipid raft fractions, suppressed caveolin-1 expression in the lipid rafts, and induced Fas translocation into lipid rafts, suggesting that simvastatin may inhibit the prosurvival PI3K/Akt pathway and trigger caspase-3-dependent apoptotic cell death through the modulation of lipid rafts. These results suggest that modulation of lipid rafts, Fas translocation, and PI3K/Akt/caspase-3 pathway are involved in the antitumor effect of simvastatin and may have a potential role in cancer prevention and treatment.

An in vitro study, evaluating the effect of sunitinib and/or lapatinib on two glioma cell lines

We sought to assess the effect of sunitinib and lapatinib applied either alone or in combination, on U87 and M059K glioma cells. Both cell lines were cultured as recommended by the manufacturer. Sunitinib and lapatinib were applied, either separately or in combination, in the cultured cells after cell attachment at doses of 10 nM, 100 nM, 1 microM and 10 microM. To determine whether the agents affect the proliferation of glioma cells, the 3-[4,5-dimethylthiazol-2-yl]-2,5 dimethyltetrazolium bromide assay was used. Apoptosis was detected using annexin V/propidium iodide detection assay, migration assay was performed in 24-well microchemotaxis chambers. The release of MMPs into the culture medium of U87 and M059K cells was measured by zymography. Both agents, administered either alone or in combination, decreased cell proliferation in a dose-dependent manner 48 h after their application in both cell lines. The inhibition of their combination was statistically different than the inhibition of each agent alone. Apoptosis was increased and migration of U87 and M059K cells was inhibited either by each agent alone or their combination. MMPs levels remained unaffected by the application of both agents in U87 cells. However, MMP-9 and MMP-2 levels were decreased 48 h after treatment of M059K cells with sunitinib either alone or in combination with lapatinib. Sunitinib and/or lapatinib appear to exhibit significant effects on proliferation, apoptosis and migration of glioma cells. When applied alone, sunitinib appears to be a more potent inhibitor than lapatinib.

The impact of conventional and heavy ion irradiation on tumor cell migration in vitro

Purpose: The influence of X-ray and 12C heavy ion irradiation on tumor cell migration and of β3 and β1 integrin expression was investigated.Material and methods: Two different tumor cell lines (U87 glioma and HCT116 colon carcinoma cells) were irradiated with 1, 3, or 10 Gy X-rays or 12C heavy ions. 24 h after irradiation a standardized Boyden Chamber assay for migration analysis was performed and cells were lysed for Western blotting.Results: Radiation-induced influences were cell line- and radiation type-dependent. X-rays decreased HCT116 migration at higher doses and appear to increase U87 migration after 3 Gy. Heavy ions decreased migration of both cell lines dose-dependently. A trend of increased β3 and β1 integrin expression in U87 cells after both radiation types was observed. β1 integrin expression in HCT116 cells was increased after X-rays but decreased after heavy ion irradiation.Conclusions: Results suggest that irradiation of tumor cells can modulate their migratory behavior. An increased migration, as shown with U87, leaves a higher probability of metastatic induction after irradiation of solid tumors in vivo, whereas an invariably reduced tumor cell migration, as shown after heavy ion treatment, could diminish the hazard of radiation-induced metastasis. As integrin expression and migration were only partially correlated, other migration-related surface molecules may be more relevant for radiation effects on tumor cell motility.