Migration Of U251 Glioma Cells Research Articles

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 μM), TMZ (155 μM), and NGF (7.55 × 10−3 μM) inhibited 43.9%–60.3%, 73.5%–81.3%, 66.2% the clonogenicity of glioma U251 cells for 1–2 days, respectively. LL-37 (4 μM), and NGF (7.55 × 10−3 μM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1–3 days. In contrast, PG-1 (16 μM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Effects of syndecan-1 on the expression of syntenin and the migration of U251 glioma cells.

Glioma is the most frequently occuring primary brain tumor. Syndecan-1 (SDC1) expression is related to poor prognosis of numerous human malignancies including glioma. Syndecan binding protein (SDCBP) is an important partner for SDC1. The present study investigated whether SDC1 and SDCBP are expressed in glioma and their functions on glioma cell migration. An immunohistochemical assay revealed that SDC1 and SDCBP were expressed and were positively related to malignant level of glioma (SDC1, rs=0.576, P=0.001; SDCBP, rs=0.661, P<0.001). Moreover, the protein levels of SDC1 were positively correlated with those of SDCBP in glioma tissues (rs=0.628, P=0.001). In U251 glioma cells, protein levels of SDC1 and SDCBP were both upregulated in U251 cells with SDC1 overexpression, while downregulated with SDC1 knockdown. Transwell assay and scratch-wound healing assay showed that SDC1 overexpression significantly increased U251 cell migration, while SDC1 knockdown had the opposite effects. Rac1 activity, signal transducer and activator of transcription 3 (STAT3) phosphorylation, as well as expression of matrix metalloproteinase 2 (MMP2) and MMP9 was significantly increased by SDC1 overexpression, while was decreased by SDC1 knockdown. In conclusion, SDC1 overexpression upregulated SDCBP expression, and promoted glioma cell migration via Rac1 activation.

Juglone reduces growth and migration of U251 glioblastoma cells and disrupts angiogenesis.

Accumulating data show that prolylisomerase (Pin1) is overexpressed in human glioblastoma multiforme (GBM) specimens. Therefore, Pin1 inhibitors should be investigated as a new chemotherapeutic drug that may enhance the clinical management of human gliomas. Recently, juglone, a Pin1 inhibitor, was shown to exhibit potent anticancer activity in various tumor cells, but its role in human glioma cells remains unknown. In the present study, we determined if juglone exerts antitumor effects in the U251 human glioma cell line and investigated its potential underlying molecular mechanisms. Cell survival, apoptosis, migration, angiogenesis and molecular targets were identified with multiple detection techniques including the MTT cell proliferation assay, dual acridine orange/ethidium bromide staining, electron microscopy, Transwell migration assay, chick chorioallantoic membrane assay, quantitative real-time polymerase chain reaction and immunoblotting. The results showed that 5–20 µM juglone markedly suppressed cell proliferation, induced apoptosis, and enhanced caspase-3 activity in U251 cells in a dose- and time-dependent manner. Moreover, juglone inhibited cell migration and the formation of new blood vessels. At the molecular level, juglone markedly suppressed Pin1 levels in a time-dependent manner. TGF-β1/Smad signaling, a critical upstream regulator of miR-21, was also suppressed by juglone. Moreover, the transient overexpression of Pin1 reversed its antitumor effects in U251 cells and inhibited juglone-mediated changes to the TGF-β1/miR-21 signaling pathway. These findings suggest that juglone inhibits cell growth by causing apoptosis, thereby inhibiting the migration of U251 glioma cells and disrupting angiogenesis; and that Pin1 is a critical target for juglones antitumor activity. The present study provides evidence that juglone has in vitro efficacy against glioma. Therefore, additional studies are warranted to examine the clinical potential of juglone in human gliomas.

Involvement of ROS-alpha v beta 3 integrin-FAK/Pyk2 in the inhibitory effect of melatonin on U251 glioma cell migration and invasion under hypoxia.

BackgroundMelatonin, a well-known antioxidant, has been shown to possess anti-invasive properties for glioma. However, little is known about the effect of melatonin on glioma cell migration and invasion under hypoxia, which is a crucial microenvironment for tumor progress. In addition, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration and invasion. Therefore, we investigated the possible role of these kinases and its related signaling in the regulation of human U251 glioma cells behavior by melatonin under hypoxia.MethodsThe abilities of migration and invasion of U251 glioma cells were determined by wound healing and transwell assay in vitro. The intracellular production of reactive oxygen species (ROS) was measured by using the fluorescent probe 6-carboxy-2′, 7′-dichorodihydrofluorescein diacetate (DCFH-DA). Immunofluorescence experiments and western blotting analysis were used to detect the expression level of protein. Small interfering RNAs (siRNA) was used to silence specific gene expression.ResultsThe pharmacologic concentration (1 mM) of melatonin significantly inhibited the migration and invasion of human U251 glioma cells under hypoxia. The inhibitory effect of melatonin was accompanied with the reduced phosphorylation of FAK and Pyk2, and decreased expression of alpha v beta 3 (αvβ3) integrin. Additionally, inhibition of αvβ3 integrin by siRNA reduced the phosphorylation of FAK/Pyk2 and demonstrated the similar anti-tumor effects as melatonin, suggesting the involvement of αvβ3 integrin- FAK/Pyk2 pathway in the anti-migratory and anti-invasive effect of melatonin. It was also found that melatonin treatment decreased the ROS levels in U251 glioma cells cultured under hypoxia. ROS inhibitor apocynin not only inhibited αvβ3 integrin expression and the phosphorylation levels of FAK and Pyk2, but also suppressed the migratory and invasive capacity of U251 glioma cells under hypoxia.ConclusionsThese results suggest that melatonin exerts anti-migratory and anti-invasive effects on glioma cells in response to hypoxia via ROS-αvβ3 integrin-FAK/Pyk2 signaling pathways. This provides evidence that melatonin may be a potential therapeutic molecule targeting the hypoxic microenvironment of glioma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0454-8) contains supplementary material, which is available to authorized users.

Effect of BMI1 Knockdown on Cell Proliferation, Apoptosis, Invasiveness, and Migration of U251 Glioma Cells

BMI1 belongs to the polycomb-repressive complex 1 (PRC1) family of genes that are conserved chromatin silencers. These are essential for maintaining both the normal and cancerous stem cell state. In this study, we evaluated the effect of siRNA-mediated BMI1 knockdown on tumor cell properties such as invasion, migration, and apoptosis, as well as on cell signaling pathways responsible for tumor progression in the human glioma cell line, U251. Knockdown of BMI1 induced apoptosis by activating cleavage of PARP and caspase-3. It also decreased the expression of anti-apoptotic proteins, survivin, XIAP, Bcl-xL, and Mcl1. Additionally, BMI1 knockdown significantly decreased cell invasion and cell migration ability. BMI1 knockdown also decreased the phosphorylation of Akt/FOXO1/3a signaling proteins. Our results suggest that BMI1 knockdown induces apoptosis and decreases cell invasion and cell migration. Moreover, we believe these phenomenona are associated with decreased phoshorylation of Akt signaling proteins, which contributes to cancer progression.