Migration Of Human Glioma Cells Research Articles

P129, a pyrazole ring-containing isolongifolanone-derivate: synthesis and investigation of anti-glioma action mechanism

BackgroundCyclin-dependent kinase-2 (CDK-2) is an important regulatory factor in the G1/S phase transition. CDK-2 targeting has been shown to suppress the viability of multiple cancers. However, the exploration and application of a CDK-2 inhibitor in the treatment of glioblastoma are sparse.MethodsWe synthesized P129 based on isolongifolanone, a natural product with anti-tumor activity. Network pharmacology analysis was conducted to predict the structural stability, affinity, and pharmacological and toxicological properties of P129. Binding analysis and CETSA verified the ability of P129 to target CDK-2. The effect of P129 on the biological behavior of glioma cells was analyzed by the cell counting kit-8, colony formation, flow cytometry, and other experiments. Western blotting was used to detect the expression changes of proteins involved in the cell cycle, cell apoptosis, and epithelial–mesenchymal transition.ResultsBioinformatics analysis and CETSA showed that P129 exhibited good intestinal absorption and blood–brain barrier penetrability together with high stability and affinity with CDK-2, with no developmental toxicity. The viability, proliferation, and migration of human glioma cells were significantly inhibited by P129 in a dose- and time-dependent manner. Flow cytometry and western blotting analyses showed G0/G1 arrest and lower CDK-2 expression in cells treated with P129 than in the controls. The apoptotic ratio of glioma cells increased significantly with increasing concentrations of P129 combined with karyopyknosis and karyorrhexis. Apoptosis occurred via the mitochondrial pathway.ConclusionThe pyrazole ring-containing isolongifolanone derivate P129 exhibited promising anti-glioma activity by targeting CDK-2 and promoting apoptosis, indicating its potential importance as a new chemotherapeutic option for glioma.

Collagen Matrices Mediate Glioma Cell Migration Induced by an Electrical Signal.

Glioma cells produce an increased amount of collagen compared with normal astrocytes. The increasing amount of collagen in the extracellular matrix (ECM) modulates the matrix structure and the mechanical properties of the microenvironment, thereby regulating tumor cell invasion. Although the regulation of tumor cell invasion mainly relies on cell–ECM interaction, the electrotaxis of tumor cells has attracted great research interest. The growth of glioma cells in a three-dimensional (3D) collagen hydrogel creates a relevant tumor physiological condition for the study of tumor cell invasion. In this study, we tested the migration of human glioma cells, fetal astrocytes, and adult astrocytes in a 3D collagen matrix with different collagen concentrations. We report that all three types of cells demonstrated higher motility in a low concentration of collagen hydrogel (3 mg/mL and 5 mg/mL) than in a high concentration of collagen hydrogel (10 mg/mL). We further show that human glioma cells grown in collagen hydrogels responded to direct current electric field (dcEF) stimulation and migrated to the anodal pole. The tumor cells altered their morphology in the gels to adapt to the anodal migration. The directedness of anodal migration shows a field strength-dependent response. EF stimulation increased the migration speed of tumor cells. This study implicates the potential role of an dcEF in glioma invasion and as a target of treatment.

Retraction.

Retraction: "Knockdown of lncRNA-UCA1 inhibits cell viability and migration of human glioma cells by miR-193a-mediated downregulation of CDK6," by Haibin Xin, Nina Liu, Xiaosheng Xu, Jinwu Zhang, Yu Li, Yongchao Ma, Guoqiang Li, Junjun Liang, J Cell Biochem. 2019; 15157-15169: The above article, published online on 20 May 2019 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/abs/10.1002/jcb.28777), has been retracted by agreement between the the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. Several flaws and inconsistencies between results presented and experimental methods described were found, the editors consider the conclusions of this article to be invalid. The authors collaborated in the investigation initially, but were not available for a final confirmation of the retraction.

Effects of SV2B on proliferation, invasion and migration of human glioma cells

Objective To study the effects of synaptic vesicle glycoprotein 2B（SV2B） on proliferation, invasion and migration of human glioma cells. Methods Bioinformatics analysis of the prognosis of malignant glioma related genes, and to establish a stable expression SV2B cell line U251 and U87, using the fluorescence microscope and image cells infected with fluorescence efficiency, further extraction cell protein and Westblot experiment, testing each SV2B protein expression, scratch test each cell migration ability, Transwell migration experiment testing each cell migration ability, Transwell invasion experiment testing each cell invasive, EdU experiment testing each cell proliferation, The cell proliferation ability was detected by plate colony formation assay. Results Bioinformatics analysis showed that SV2B was a gene associated with poor prognosis in glioma, and high expression of SV2B protein was associated with shorter survival time in GBM patients. In the stably expressing SV2B cell lines constructed by lentivirus, overexpression of SV2B family caused the enhancement of proliferation, invasion and migration of human glioma cells. Conclusion SV2B plays an important role in proliferation, invasion and migration of human glioma cells.

Rap2B promotes the proliferation and migration of human glioma cells via activation of the ERK pathway.

Glioma is one of the most common primary brain tumors and has a poor prognosis. Rap2B, a member of the Ras family of oncogenes, is highly expressed and promotes the progression of several tumors, including glioma. However, the mechanism underlying the role of Rap2B in glioma is not fully understood. In the present study, after transfection, Rap2B expression was detected by reverse transcription PCR and western blot analysis. Cell proliferation and cell migration assays were performed to determine the effects of Rap2B on the malignant biological behaviors of glioma cells. The changes of ERK pathway-associated proteins were examined by western blot analysis. Enzyme-linked immunosorbent assay (ELISA) and western blot analysis were utilized to detect the protein levels of matrix metalloproteinase (MMP)2 and MMP9. Then, The Cancer Genome Atlas database was used to determine the association between Rap2B expression and clinical parameters in patients with glioblastoma multiforme and low-grade glioma (LGG). Results revealed that Rap2B was highly expressed in human glioma compared with that in adjacent normal tissues and normal human astrocytes, and that silenced Rap2B led to a reduction of cell proliferation and migration ability in glioma cells. Conversely, overexpressed Rap2B in both U87 and U251 cells significantly enhanced these malignant activities. In addition, ELISA assay and western blotting showed that Rap2B increased MMP2 and MMP9 expression. The western blot assay revealed that Rap2B induced the phosphorylation of ERK in glioma cells. Furthermore, silencing the ERK pathway by SCH772984 led to the inhibition of Rap2B-mediated proliferation, migration and the reduction of MMP2 and MMP9 expression. Kaplan-Meier analysis revealed that increased Rap2B expression was associated with poorer survival of patients with LGG. These results demonstrated that Rap2B may participate in the processes of glioma cell proliferation and migration through enhancing MMP2 and MMP9 expression via the ERK pathway. Thus, Rap2B could potentially be used as a promising therapeutic target and prognostic biomarker in glioma.

γ-secretase inhibitors, DAPT and RO4929097, promote the migration of Human Glioma Cells via Smad5-downregulated E-cadherin Expression.

Malignant gliomas are a type of central nervous system cancer with extremely high mortality rates in humans. γ-secretase has been becoming a potential target for cancer therapy, including glioma, because of the involvement of its enzymatic activity in regulating the proliferation and metastasis of cancer cells. In this study, we attempted to determine whether γ-secretase activity regulates E-cadherin to affect glioma cell migration. The human glioma cell lines, including LN18 and LN229, and the γ-secretase inhibitors, including N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and RO4929097, were used in this study. It was shown that γ-secretase activity inhibition by DAPT and RO4929097 could promote LN18 and LN229 glioma cell migration via downregulating E-cadherin mRNA and protein expressions, but not via affecting E-cadherin protein processing. In addition, γ-secretase activity inhibition was regulated by bone morphogenetic proteins-independent Smad5 activation in glioma cells. Moreover, endogenous Smad1 in glioma cells was found to play an important role in regulating E-cadherin expression and subsequent cell migration but did not affect DAPT-stimulated effects. These results help further elucidate the molecular mechanisms of γ-secretase activity regulation involved in controlling glioma cell malignancy. Information about a potential role for Smad1/5 activity upregulation and subsequent E-cadherin downregulation during inhibition of γ-secretase activity in the development of gliomas is therefore relevant for future research.

Neuregulin 2 (NRG2) is expressed in gliomas and promotes migration of human glioma cells.

Glioma is the most common primary brain tumour in adults. Numerous studies have shown that neuregulins (NRGs) may be involved in the formation of glioma. Although NRG1 has been extensively studied in glioma, the functions of NRG2 in glioma development remain elusive. In the present study, we investigated the expression of NRG2 in different grades of human glioma samples, and analysed the functional effects of NRG2 in glioma cells mainly using wound healing assay and transmigration assay. We found that NRG2 was differentially expressed in different grades of human glioma/glioblastoma tissues. The data from wound healing assays demonstrated that NRG2 can differentially promote the migration of SHG44 human glioma, and U251 and U-87 MG human glioblastoma cells at different time points. The results of cell transmigration assays showed that, compared with the vehicle control, the number of cells that migrated to the underside of the insert was increased significantly for all the 3 cell lines treated with 5 nM of NRG2 for 12 hours. In conclusion, our results demonstrated that NRG2 is expressed in gliomas to varying extents, and it may play roles in the migration of glioma cells in vitro. These data suggest that treatment targeting NRG2 signalling may partly reverse the migration-based metastasis of glioma cells.

ACTL6A knockdown inhibits cell migration by suppressing the AKT signaling pathway and enhances the sensitivity of glioma cells to temozolomide.

Molecular-targeted therapy has had a significant impact on glioma. Notably, actin-like 6A (ACTL6A) has been indicated to be essential for embryonic development and tumor progression. However, the role of ACTL6A in glioma remains unclear. The present study aimed to investigate the effects of ACTL6A on glioma cell migration and sensitivity to temozolomide (TMZ). The expression levels of ACTL6A were analyzed in patients with glioma, and survival curves were created using data from The Cancer Genome Atlas. U251 and T98G cells were transfected with short hairpin (sh)RNA for use in loss-of-function experiments to investigate the biological function and molecular mechanisms of ACTL6A. Furthermore, an MTT assay was used to assess the effect of ACTL6A on the sensitivity of glioma cells to TMZ. The results demonstrated that ACTL6A was expressed at higher levels in glioma tissues compared with normal brain tissues. Furthermore, high expression of ACTL6A was associated with a poor prognosis. The knockdown of ACTL6A significantly inhibited the migration phenotype in glioma cells and significantly decreased the levels of phosphorylated AKT in glioma cells. The AKT signaling activator SC79 partly attenuated the inhibitory effects of ACTL6A shRNA on glioma cell migration. Additionally, the knockdown of ACTL6A enhanced the sensitivity of glioma cells to TMZ. In conclusion, these results suggest that ACTL6A knockdown inhibited the migration of human glioma cells, at least in part through inactivation of the AKT signaling pathway, and increased the sensitivity of glioma cells to TMZ. Therefore, ACTL6A may be a potential therapeutic target for glioma.

PI3K/mTORC1/2 inhibitor PQR309 inhibits proliferation and induces apoptosis in human glioblastoma cells.

Glioblastoma (GBM) is the most common type of primary central nervous system tumor in adults, which has high mortality and morbidity rates, and short survival time, namely <15 months after the diagnosis and application of standard therapy, which includes surgery, radiation therapy and chemotherapy; thus, novel therapeutic strategies are imperative. The activation of the PI3K/AKT signaling pathway plays an important role in GBM. In the present study, U87 and U251 GBM cells were treated with the PI3K/mTORC1/2 inhibitor PQR309, and its effect on glioma cells was investigated. Cell Counting Kit-8 assay, 5-ethynyl-2′-deoxyuridine and colony formation assays revealed dose- and time-dependent cytotoxicity in glioma cells that were treated with PQR309. Flow cytometry and western blotting revealed that PQR309 can significantly induce tumor cell apoptosis and arrest the cell cycle in the G1 phase. Furthermore, the expression levels of AKT, phosphorylated (p)-AKT, Bcl-2, Bcl-xL, Bad, Bax, cyclin D1, cleaved caspase-3, MMP-9 and MMP-2 were altered. In addition, the migration and invasion of glioma cells, as detected by wound healing, migration and Transwell invasion assays, exhibited a marked suppression after treating the cells with PQR309. These results indicated that PQR309 exerts an antitumor effect by inhibiting proliferation, inducing apoptosis, inducing G1 cell cycle arrest, and inhibiting invasion and migration in human glioma cells. The present study provides evidence supportive of further development of PQR309 for adjuvant therapy of GBM.

TRPM7 Induces Mechanistic Target of Rap1b Through the Downregulation of miR-28-5p in Glioma Proliferation and Invasion.

Objectives: Our previous findings demonstrate that channel-kinase transient receptor potential (TRP) ion channel subfamily M, member 7 (TRPM7) is critical in regulating human glioma cell migration and invasion. Since microRNAs (miRNAs) participate in complex regulatory networks that may affect almost every cellular and molecular process during glioma formation and progression, we explored the role of miRNAs in human glioma progression by comparing miRNA expression profiles due to differentially expressed TRPM7.Methods: First, we performed miRNA microarray analysis to determine TRPM7's miRNA targets upon TRPM7 silencing in A172 cells and validated the miRNA microarray data using A172, U87MG, U373MG, and SNB19 cell lines by stem-loop RT-qPCRs. We next determined whether TRPM7 regulates glioma cell proliferation and migration/invasion through different functional domains by overexpressing wild-type human TRPM7 (wtTRPM7), two mutants with TRPM7's α-kinase domain deleted (Δkinase-DK), or a point mutation in the ATP binding site of the α-kinase domain (K1648R-KR). In addition, we determined the roles of miR-28-5p in glioma cell proliferation and invasion by overexpressing or under expressing miR-28-5p in vitro. Lastly, we determined whether a Ras-related small GTP-binding protein (Rap1b) is a target of miR-28-5p in glioma tumorigenesis.Results: The miRNA microarray data revealed a list of 16 downregulated and 10 upregulated miRNAs whose transcripts are significantly changed by TRPM7 knock-down. Cell invasion was significantly reduced in two TRPM7 mutants with inactive kinase domain, Δkinase, and K1648R transfected glioma cells. miR-28-5p overexpression suppressed glioma cells' proliferation and invasion, and miR-28-5p under expression led to a significant increase in glioma cell proliferation and migration/invasion compared to that of the controls. miR-28-5p suppressed glioma cell proliferation and migration by targeting Rap1b. Co-transfection of siRap1b with miR28-5p inhibitor reduced the glioma cell proliferation and invasion, caused by the latter.Conclusions: These results indicate that TRPM7's channel activity is required for glioma cell growth while the kinase domain is required for cell migration/invasion. TRPM7 regulates miR-28-5p expression, which suppresses cell proliferation and invasion in glioma cells by targeting Rap1b signaling.

Curcumin suppresses invasiveness and migration of human glioma cells in vitro by inhibiting HDGF/β-catenin complex

To investigate the effect of curcumin on the invasion and migration of human glioma cells in vitro and explore the molecular mechanisms. MTT assay was used for screening the optimal curcumin concentrations. The effects of curcumin on the invasion and metastasis of human glioma cell lines U251 and LN229 were tested using Transwell assay, Boyden assay and wound-healing assays. The expression of the related proteins and their interactions were determined using Western blotting and coimmunoprecipitation assay. Curcumin at the concentration of 20 μmol/L for 48 h was used as the optimal condition for subsequent cell treatment. In the two glioma cell lines, curcumin significantly suppressed the invasion and migration of the cells (P &lt; 0.05) and lowered the expressions of hepatoma-derived growth factor (HDGF), Ncadherin, vimentin, Snail and Slug, but increased the expression of E-cadherin. Interference of HDGF in curcumin-treated glioma cells synergistically inhibited the epithelial-mesenchymal transition (EMT) signals, while overexpression of HDGF significantly reversed the inhibitory effect of curcumin on EMT; curcumin treatment could significantly reduce the binding of HDGF to β-catenin. Curcumin suppresses EMT signal by reducing HDGF/β-catenin complex and thereby lowers the migration and invasion abilities of human glioma cells in vitro.

Knockdown of lncRNA-UCA1 inhibits cell viability and migration of human glioma cells by miR-193a-mediated downregulation of CDK6.

Long noncoding RNA urothelial carcinoma-associated 1 (lncRNA-UCA1) is generally recognized as an oncogenic molecule in several human malignant tumors. However, the available evidence does not necessarily imply an unequivocal causal function of UCA1 in glioblastoma. The current study was aimed to probe the biological function of lncRNA-UCA1 in human glioblastoma cell lines. Besides, we further investigated the potential mechanisms. Cell viability, apoptosis, as well as migration and invasion were measured using a commercial cell counting kit-8, flow cytometry, and 24-Transwell assay, respectively. LncRNA-UCA1, microRNA-193a (miR-193a), and CDK6 at messenger RNA levels were evaluated by quantitative real-time polymerase chain reaction method. Protein level was examined by Western blot analysis. RNA immunoprecipitation was utilized to validate lncRNA-UCA1 associated with miR-193a. Luciferase activity assay was used to identify the miR-193a-targeted CDK6 3'-untranslated region. lncRNA-UCA1 knockdown weakened cell viability, augmented apoptosis progression, as well as suppressed migration and invasion behaviors in glioblastoma cells, whereas lncRNA-UCA1 silence exhibited the opposite functions. lncRNA-UCA1 functioned as an endogenous sponge of miR-193a. miR-193a silence reversed the biological function of lncRNA-UCA1 knockdown on U-118 MG cells. miR-193a negatively regulated the expression of CDK6, and it affected the U-118 MG cells through regulating CDK6 expression. CDK6 overexpression abrogated the blockage of PI3K/AKT, mitogen-activated protein kinase (MAPK), and Notch signaling pathways. Furthermore, lncRNA-UCA1 and miR-193a could affect these signaling cascades through regulating CDK6 expression. The regulatory mechanisms of lncRNA-UCA1 were further consolidated in clinical specimens. lncRNA-UCA1 silence reduced cell viability, promoted apoptosis progression, while impeding the migration and invasion of glioblastoma cells by miR-193a-mediated silence of CDK6, with blockage of PI3K/AKT, MAPK, and Notch pathways.

MicroRNA‑34a inhibits cell growth and migration in human glioma cells via MMP‑9.

The present study was designed to investigate the function of matrix metalloproteinase-9 (MMP-9) in human glioma cells and the potential regulatory mechanisms. Reverse transcription-quantitative polymerase chain reaction was used to analyze the expression of MMP-9 and microRNA-34a (miR-34a) in the plasma of patients with glioma and healthy volunteers. MTT and Transwell assays were used to assess cell growth and migration, respectively. Annexin-V/propidium iodide staining was used to measure cell apoptosis. In addition, MMP-9 expression was measured using western blot analysis. In patients with glioma, MMP-9 expression was increased, while miR-34a expression was suppressed, compared with the normal group. Overall survival (OS) and disease-free survival (DFS) of patients with high MMP-9 expression were decreased compared with those with low MMP-9 expression. OS and DFS of patients with low miR-34a expression were decreased compared with those with high miR-34a expression. Downregulation of miR-34a promoted cell growth and migration, and inhibited apoptosis in U251-MG glioma cells. However, overexpression of miR-34a inhibited cell growth and migration, and induced apoptosis in glioma cells. Furthermore, downregulation of miR-34a using anti-miR-34a induced MMP-9 protein expression in glioma cells; whereas, overexpression of miR-34a suppressed MMP-9 protein expression in glioma cells. SB-3CT, an inhibitor of MMP-9, attenuated the effects of miR-34a mimic on glioma cells. Together, these results indicated that miR-34a inhibited cell growth and migration in human glioma cells by regulating MMP-9.

MiRNA-139–3p inhibits the proliferation, invasion, and migration of human glioma cells by targeting MDA-9/syntenin

Gliomas are the most common primary malignant brain tumor in adults. Although these tumors are aggressive and frequently lethal, there are currently few therapeutic approaches available to prolong patient survival. MicroRNAs play important roles in regulating the expression of genes that control diverse cellular processes. Here, we investigated the expression and function of miR-139–3p in gliomas using clinical specimens, cultured cells, and a mouse xenograft tumor model. We found that miR-139–3p expression is markedly lower in human glioma tissues than in normal brain tissues. We identified melanoma differentiation-associated gene-9 (MDA-9)/syntenin, an adaptor protein implicated in tumor metastasis, as a novel direct target of miR-139–3p and showed that syntenin mRNA and miR-139–3p levels were inversely correlated in clinical specimens (r = −0.6817, P = 0.0002). Overexpression of miR-139–3p in human glioma cell lines inhibited cell proliferation, migration, and invasion, and these effects were rescued by co-transfection with syntenin. Our results indicate that miR-139–3p plays a significant role in controlling behaviors associated with the malignant progression of gliomas, and we identify the miR-139-3p–syntenin axis as a potential therapeutic target for glioma.

Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

Regulatory effect of annexin A2 on invasion and migration of human glioma cells

Objective To investigate the regulatory effect of annexin A2 (ANXA2) on invasion and migration of human glioma cells. Methods The ANXA2 small interfering RNA (siRNA) and siRNA control were transfected into glioma cells U87 and labeled as ANXA2 siRNA group and siRNA-NC group. The control group was set up at the same time. The expression of ANXA2 mRNA and protein was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively. Cell proliferation was detected by methyl thiazol tetrazolium (MTT). The cell migration and invasive ability was detected by Transwell. Western blotting was used to detect E-cadherin and matrix metalloproteinase (MMP)-2 proteins. Results The levels of ANXA2 mRNA (0.85±0.08, P=0.258) and protein (0.72±0.09, P=0.751) in siRNA-NC group were not statistically differed from those in the control group (for mRNA and protein: 0.87±0.05 and 0.75±0.07). The survival rate [(98.87±11.74)%, P=0.695], the number of invasive cells (40.65±4.85, P=0.410), the number of migrating cells (66.87±7.64, P=0.278) and the levels of E-cadherin (0.70±0.09, P=0.320) and MMP-2 protein (0.29±0.02, P=0.196) in siRNA-NC group showed no statistically significant difference from those in the control group. The survival rate [(52.98±5.61)%, P=0.006], the number of invasive cells (23.22±2.25, P=0.002), the number of migrating cells (41.68±3.87, P=0.002) and the levels of MMP-2 protein (0.11±0.03, P=0.001) in the ANXA2 siRNA group were significantly lower, and the levels of E-cadherin protein (1.14±0.11, P=0.002) were significantly higher than those in the control group (0.72±0.06). Conclusion Interference with ANXA2 can inhibit the proliferation, invasion and migration of glioma cells, reduce the expression level of MMP-2 and promote the expression of E-cadherin. Key words: Glioma; Invasion; Annexin A2; Migration

Involvement of P2X7 Receptor in Proliferation and Migration of Human Glioma Cells.

Previous studies have demonstrated that activation of P2X7 receptors (P2X7R) results in the proliferation and migration of some types of tumor. Here, we asked whether and how the activated P2X7R contribute to proliferation and migration of human glioma cells. Results showed that the number of P2X7R positive cells was increasing with grade of tumor. In U87 and U251 human glioma cell lines, both expressed P2X7R and the expression was enhanced by 3′-O-(4-benzoylbenzoyl) ATP (BzATP), the agonist of P2X7R, and siRNA. Our results also showed that 10 μM BzATP was sufficient to induce the proliferation of glioma cell significantly, while the cell proliferation reached the peak with 100 μM BzATP. Also, the migration of U87 and U251 cells was significantly increased upon BzATP treatment. However, the number of apoptotic cells of U87 and U251 was not significantly changed by BzATP. In addition, the expression of ERK, p-ERK, and proliferating cell nuclear antigen (PCNA) protein was increased in BzATP-treated U87 and U251 glioma cells. PD98059, an inhibitor of the MEK/ERK pathway, blocked the increased proliferation and migration of glioma cells activated by BzATP. These results suggest that ERK pathway is involved in the proliferation and migration of glioma cells induced by P2X7R activation.

Ubiquitin carboxyl-terminal hydrolase isozyme L5 inhibits human glioma cell migration and invasion via downregulating SNRPF.

Ubiquitin C-terminal Hydrolase-L5 (UCH-L5/UCH37), a member of the deubiquitinases (DUBs), suppresses protein degeneration via removing ubiquitin from the distal subunit of the polyubiquitin chain. The activity of UCH-L5 is enhanced when UCH-L5 combines with proteasome 19S regulatory subunit by Rpn13/Admr1 receptor and inhibited when UCH-L5 interacts with NFRKB. But the role of UCH-L5 in gliomas remains unknown. In this study, analysis of 19 frozen and 51 paraffin-embedded clinic pathological cases showed that UCH-L5 expression in glioma tissues was lower than normal brain tissues. In vitro, we found that UCH-L5 could inhibit migration and invasion of U87MG and U251 cells. It has been reported that the expression of SNRPN, SNRPF, and CKLF was abnormal in gliomas or other tumors. We also found that SNRPF-siRNA, SNRPN-siRNA and CKLF-siRNA could inhibit migration and invasion of U87MG cells. And knockdown of UCH-L5 expression improved both mRNA expression and protein level of SNRPF. The relationship between UCH-L5 and SNRPF was further confirmed in 293T cells. Our study showed that UCH-L5 could inhibit migration and invasion of glioma cells via down regulating expression of SNRPF. And the above findings suggest that UCH-L5 may inhibit occurrence and metastasis of gliomas.

CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells.

The cell adhesion molecule with homology to L1CAM (close homolog of L1) (CHL1) is a member of the cell adhesion molecule L1 (L1CAM) gene family. Although CHL1 expression and function have been reported in several tumors, the roles of CHL1 in the development of glioma remain unclear. In the present study, we investigated the effects of CHL1 on proliferation indexes and activation of Akt1 and Erk signaling by siRNA in U-87 MG human glioblastoma and human U251 and SHG-44 glioma cells. We found that siRNA targeting CHL1 significantly down-regulated the expression of CHL1 mRNA and protein accompanied by reduced cell proliferation and transmigration invasion in all three cell lines. Down-regulating CHL1 expression also reduced cell survival, as measured by the Bax/Bcl-2 ratio, and increased activation of caspase-3. In subcutaneous U-87 MG cell xenograft tumors in nude mice, intratumoral administration of siRNA targeting CHL1 treatment significantly down-regulated CHL1 expression in vivo, accompanied by increased levels of activated caspase-3. Our combined results confirmed for the first time that in contrast to findings about CHL1 in most other cancer types, CHL1 functions in promoting cell proliferation, metastasis and migration in human glioma cells both in vitro and in vivo. These results indicate that CHL1 is a therapeutic target in the clinical management of glioma/glioblastoma.

Abstract 900: Tumor Treating Fields (TTFields) affect human glioma cell migration, invasion and adherence properties in vitro

Abstract TTFields, an approved treatment modality for glioblastoma, are delivered via continuous, noninvasive application of low intensity, intermediate frequency alternating electric fields. The antimitotic effects of TTFields have been extensively investigated. We investigated other processes that may be affected by TTFields such as human glioma cell migration and cellular invasion properties. Four human glioma cell lines were treated with TTFields using the inovitro system. Cell migration rates were measured using wound healing assays. Invasion assays were performed using a modified Matrigel coated Boyden chamber. Cell adhesion assays were performed during TTFields treatment and compared to untreated controls. At the end of TTFields’ treatment, adhered cells were trypsinized and counted. A cells de-adhesion assay was performed following 24 and 72 hours of TTFields application with the outcome being the number of cells removed after varying times of trypsinization. Application of TTFields in-vitro led to a significant reduction in cell migration velocity compared with untreated control cells. Furthermore, comparison of the anti-migratory efficacy of TTFields applied from 2 directions vs. a single direction revealed that the single direction approach applied perpendicularly to the course of migration was more effective than TTFields applied in 2 directions or in a single parallel direction. Glioma cells invasion was significantly reduced compared to untreated cells in all tested cell lines. Cell adhesion to the substrate (fibronectin) was significantly reduced when exposed to TTFields. On the other hand, cell de-adherence following TTFields treatment took significantly longer time of trypsinization. Our results suggest that human glioma cell motility is impaired by exposure to TTFields. Both alterations in cells adherence and de-adherence during exposure to TTFields may contribute to reduction in cell motility. Further studies are needed to elucidate the mechanism by which TTFields disrupts cellular motility in glioma cancer cells. Citation Format: Dario Garcia-Carracedo, Rosa S. Schneiderman, Einav Zeevi, Anna Shteingauz2, Moshe Giladi, Tali Voloshin, Yaara Porat, Mijal Munster, Roni Blat, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) affect human glioma cell migration, invasion and adherence properties in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 900. doi:10.1158/1538-7445.AM2017-900