U87 Migration Research Articles

Cadherin-6 is a novel mediator for the migration of mesenchymal stem cells to glioblastoma cells in response to stromal cell-derived factor-1.

Glioblastoma recruits various nontransformed cells from distant tissues. Although bone marrow-derived mesenchymal stem cells (MSCs) have been observed migrating to glioblastoma, the underlying mechanism driving MSC migration toward glioblastoma remains unclear. Tumor vascularity is critical in the context of recurrent glioblastoma and is closely linked to the expression of stromal cell-derived factor-1 (SDF-1). We demonstrated that cadherin-6 mediated MSC migration both toward SDF-1 and toward glioblastoma cells. Cadherin-6 knockdown resulted in the downregulation of MSCs capacity to migrate in response to SDF-1. Furthermore, MSCs with cadherin-6 knockdown exhibited impaired migration in response to conditioned media derived from glioblastoma cell lines (U87 and U373) expressing SDF-1, thus simulating the glioblastoma microenvironment. Moreover, MSCs enhanced the vasculogenic capacity of U87 cells without increasing the proliferation, cancer stem cell characteristics, or migration of U87. These results suggest that the current strategy of utilizing MSCs as carriers for antiglioblastoma drugs requires careful examination. Furthermore, cadherin-6 may represent a novel potential target for controlling the recruitment of MSCs toward glioblastoma.

RND1 inhibits epithelial-mesenchymal transition and temozolomide resistance of glioblastoma via AKT/GSK3-β pathway

ABSTRACT GBM is one of the most malignant tumor in central nervous system. The resistance to temozolomide (TMZ) is inevitable in GBM and the characterization of TMZ resistance seriously hinders clinical treatment. It is worthwhile exploring the underlying mechanism of aggressive invasion and TMZ resistance in GBM treatment. Bioinformatic analysis was used to analyze the association between RND1 and a series of EMT-related genes. Colony formation assay and cell viability assay were used to assess the growth of U87 and U251 cells. The cell invasion status was evaluated based on transwell and wound-healing assays. Western blot was used to detect the protein expression in GBM cells. Treatment targeted RND1 combined with TMZ therapy was conducted in nude mice to evaluate the potential application of RND1 as a clinical target for GBM. The overexpression of RND1 suppressed the progression and migration of U87 and U251 cells. RND1 knockdown facilitated the growth and invasion of GBM cells. RND1 regulated the EMT of GBM cells via inhibiting the phosphorylation of AKT and GSK3-β. The promoted effects of RND1 on TMZ sensitivity was identified both in vitro and in vivo. This research demonstrated that the overexpression of RND1 suppressed the migration and EMT status by downregulating AKT/GSK3-β pathway in GBM. RND1 enhanced the TMZ sensitivity of GBM cells both in vitro and in vivo. Our findings may contribute to the targeted therapy for GBM and the understanding of mechanisms of TMZ resistance in GBM.

VSIG4 Silencing Inhibits Glioblastoma Growth by Regulating the JAK2/STAT3 Pathway.

Glioblastoma (GBM) is the most common malignant primary brain tumour in adults. VSIG4 has been identified to be associated with GBM. We aimed to determine the downstream regulatory mechanisms of VSIG4 in GBM. Differential expression of VSIG4 was analysed using GEPIA. The expression of VSIG4 was assessed by RT-qPCR and its downstream genes were screened by transcriptome sequencing. The expression of pyroptosis-related proteins and the JAK2/STAT3 pathway was measured by Western blotting. GBM cell viability, migration, and invasion were detected using CCK-8, scratch, and Transwell assays. The levels of pyroptosis-related factors were measured using ELISA. The effect of VSIG4 on GBM tumour growth in vivo was explored by constructing a xenograft tumour model. VSIG4 expression was upregulated in GBM. Functionally, silencing of VSIG4 inhibited proliferation, invasion, and migration of U251 and LN229 cells, and promoted pyroptosis. Mechanically, transcriptome sequencing revealed that the JAK2/STAT3 pathway might be a downstream regulator of VSIG4. Further studies proved that silencing of VSIG4 enhanced the expression of p-JAK2 and p-STAT3, and the JAK2/STAT3 pathway inhibitor relieved the suppression of VSIG4 silencing on GBM cell viability, invasion, and migration. Furthermore, in vivo experiments further validated that knockdown of VSIG4 inhibited the growth of GBM tumors. In GBM, silencing VSIG4 promoted pyroptosis and inhibited tumor progression by regulating the JAK2/STAT3 signaling pathway.

CMTM6 knockdown prevents glioma progression by inactivating the mTOR pathway.

BackgroundGliomas in the adult brain are complicated and aggressive with a poor prognosis. Gene therapy is a recent alternative glioma treatment. We sought to explore the mechanism of chemokine-like factor (CKLF) MARVEL transmembrane domain-containing 6 (CMTM6) in glioma.MethodsThe Cancer Genome Atlas database reports that CMTM6 is expressed in tumors and glioma tissue. CMTM6 expression in glioma tissues and cells was detected and its relationship with clinical pathology was analyzed. Short hairpin ribonucleic acid-CMTM6 lentivirus was transfected into U87 and U251 cells to evaluate malignant glioma cells. Using the biological website (https://string-db.org/cgi/input.pl?Sessionid) and reference retrieval, the pathway that interacted with CMTM6 and related to glioma was identified. The level of the mammalian target of rapamycin pathway-related proteins was detected. Functional rescue experiments were performed using the combination of mTOR activator MHY1485 and the knockdown CMTM6. The growth of xenograft tumors was observed and Ki67-positive expression was determined.ResultsCMTM6 upregulation in gliomas was associated with a poor prognosis. CMTM6 expression was notably higher in gliomas. After the knockdown of CMTM6, the proliferation, invasion, and migration of U87 and U251 cells were inhibited, and the apoptosis rate was increased. Knocking down CMTM6 inactivated the mTOR pathway. The activation of mTOR pathway reversed the inhibitory effects of CMTM6 knockdown on glioma cell behaviors. CMTM6 knockdown reduced tumor volume, body mass, and Ki67-positive expression.ConclusionsThe knockdown of CMTM6 inhibited the activation of mTOR pathway and prevented the malignant episodes of glioma cells.

Targeting DDX3X Helicase Activity with BA103 Shows Promising Therapeutic Effects in Preclinical Glioblastoma Models.

Simple SummaryIn the last ten years, the human helicase protein DDX3X turned out to be an extremely interesting target for the development of potential anticancer drugs. Herein, we discovered BA103, a novel specific inhibitor of the helicase binding site of DDX3X, which is characterized by broad-spectrum anticancer activity. BA103 revealed promising tolerability in fibroblasts and good pharmacokinetic properties. Furthermore, BA103 was able to decrease the expression of β-catenin and to reduce tumor migration. Its capability to pass the blood–brain barrier led us to investigate its potential against glioblastoma, which is a high refractory disease with poor prognosis. High efficacy was proven in both xenograft and orthotopic animal models.DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

Downregulation of Tim-1 inhibits the proliferation, migration and invasion of glioblastoma cells via the miR-133a/TGFBR1 axis and the restriction of Wnt/\u03b2-catenin pathway

BackgroundGlioblastoma remains one of the most lethal brain cancers. T-cell immunoglobulin and mucin domain 1 (Tim-1) is associated with various immune diseases. The molecular mechanism of Tim-1 in regulating glioblastoma cell proliferation, invasion, and migration is still unknown. Moreover, it has shown that miR-133a plays an important role in glioblastoma. However, little is known about the interaction between Tim-1 and miR-133a in glioblastoma.MethodsTim-1 expression in glioblastoma and normal brain tissues was detected by qPCR, Western Blot and IHC. After Tim-1 knockdown in U251 and U87 cells, genes showing significantly differential expression, along with the significant differential miRNAs were analyzed using RNA-seq analysis. The binding sites were verified using dual-luciferase reporter gene assay. U251 and U87 cells were allocated into the small harpin-negative control (sh-NC), sh-Tim-1, sh-Tim-1 + inhibitor NC, and sh-Tim-1 + miR-133a inhibitor group. Cell proliferation, migration, and invasion were determined by CCK-8, flow cytometry, wound-healing and Transwell assays, respectively. Next, U251 and U87 cells were allocated into the mimic NC, miR-133a mimic, miR-133a mimic + pcDNA3.1, and miR-133a mimic + pcDNA3.1-TGFBR1 groups, followed by the detection of cell proliferation, migration, and invasion. Western blot was used to identify the expression of vital kinases in the Wnt/β-catenin pathway.ResultsTim-1 was highly expressed in glioblastoma tissues compared with that in normal brain tissues. RNA-seq analysis showed that Tim-1 knockdown could lead to the downregulation of TGFBR1 and the upregulation of miR-133a. The binding sites between TGFBR1 and miR-133a were confirmed. Tim-1 knockdown impaired the invasion, migration, proliferation of U251 and U87 cells, which could be reversed by miR-133a downregulation. miR-133a upregulation inhibited the proliferation, invasion, and migration of U251 and U87 cells, which could be reversed by TGFBR1 upregulation. Tim-1 knockdown and miR-133a upregulation could inhibit the activation of the Wnt/β-catenin pathway, while the elevation of TGFBR1 showed opposite effects.ConclusionTim-1 knockdown inhibited glioblastoma cell proliferation, invasion, and migration through the miR-133a/TGFBR1 axis and restrained the activation of the Wnt/β-catenin pathway.

NGMA-2. Dual sgRNA-directed PD-L1 knockout in human glioblastoma cells using the CRISPR/Cas9 system

Glioblastoma multiforme (GBM) is an astrocyte derived brain tumor. It induces an immunosuppressive microenvironment by exploiting immune checkpoints such as the PD-1/PD-L1 pathway. Targeting the PD-1/PD-L1 pathway for immunotherapy is a promising new avenue for treating GBM, but more work is needed to develop a safe and effective method for clinical applications. We identified two sgRNA sequences located on PD-L1 exon 3. The first sgRNA recognized the forward strand of human PD-L1 near the beginning of exon 3 and cuts at approximately base pair 82 (g82). The second sgRNA recognized the reverse strand of exon 3 and cuts at base pair 165 (g165). Two sgRNAs, g82 and g165, created an 83bp deletion in the genomic sequence that can lead to the production of a non-functional PD-L1 protein. A homology-directed repair template (HDR) containing an in-frame stop codon was designed to enhance PD-L1 knockout specificity and efficiency. Both g82 and g165 were cloned into the CRISPR/Cas9 plasmid, and was co-transfected with the added HDR template. T7E1, qRT-PCR and western blot analysis determined that the dual sgRNA CRISPR/Ca9 system knocked out both endogenous (80%) and exogenous (64%) PD-L1 in U87 cells and PD-L1 overexpression U87 cells, respectively. Deletion of PD-L1 reduced U87 migration and proliferation, while PD-L1 overexpression promoted tumor growth and tumor-associated macrophage polarization. Together, deletion of both membrane and cytoplasmic PD-L1 altered the PD-L1-associated immunosuppressive environment and prevented tumor progression and migration. Thus, a dual sgRNA CRISPR/Cas9 gene-editing system is a promising avenue for anti-GBM immunotherapy.

Sevoflurane inhibits cell proliferation and migration of glioma by targeting the miR‑27b/VEGF axis.

Poor prognosis in patients with glioma is primarily due to rapid tumor growth and cell invasion and migration. In addition, microRNA (miR)-27b is decreased in metastatic glioma. The present study investigated whether sevoflurane inhibited glioma cell progression by targeting miR-27b. Cell proliferation was analyzed using a Cell Counting Kit-8 assay and a wound healing assay was used to detect cell migration. Western blotting and reverse transcription-quantitative PCR analysis were performed to determine the protein and mRNA expression levels. A dual luciferase assay was used to determine the relationship between vascular epithelial growth factor (VEGF) and miR-27b. VEGF was identified to be a direct target of miR-27b. Moreover, sevoflurane treatment increased the expression of miR-27b and decreased the expression of VEGF in U251 and U87 cells. Compared with the control group, sevoflurane inhibited the proliferation and migration of U251 and U87 cells, as well as the expression of matrix metalloproteinase (MMP)-2 and MMP-9, which were subsequently abolished by pre-treatment with an miR-27b inhibitor. The present results indicated the potential use of sevoflurane by anesthesiologists for the surgical resection of glioma, which may improve patient outcomes in the clinical setting.

LINC00210 exerts oncogenic roles in glioma by sponging miR-328.

Long non-coding RNAs (lncRNAs) have been reported to serve key roles in human cancer types, including glioma. However, to the best of our knowledge, the expression and function of lncRNA LINC00210 in glioma have not previously been investigated. The present study was conducted to explore the regulatory role of LINC00210 in glioma cells. The present study demonstrated that LINC00210 was significantly upregulated in glioma tissues, and high expression of LINC00210 was significantly associated with advanced clinical stage and poor prognosis in patients with glioma. It was found that LINC00210 knockdown significantly inhibited the proliferation and migration of U251 and T98G cells. The results of luciferase reporter assays indicated that LINC00210 could directly target microRNA (miR)-328 in glioma cells, and miR-328 expression was negatively correlated with LINC00210 expression in glioma tissues. LINC00210 knockdown significantly promoted the expression of miR-328 in U251 and T98G cells. Moreover, silencing miR-328 impaired the inhibitory effects of LINC00210 knockdown on the proliferation and migration of U251 and T98G cells. Therefore, the present results suggested that LINC00210 may exert an oncogenic role in glioma via sponging miR-328.

MiR-920 and LSP1 co-regulate the growth and migration of glioblastoma cells by modulation of JAK2/STAT5 pathway.

This study probes the function and mechanism of lymphocyte-specific protein 1 (LSP1) in glioblastoma pathogenesis. According to the data acquired from TCGA, Oncomine and GEO databases, the expression and prognostic value of LSP1 and miR-920 in glioblastoma patients were analyzed. The expression levels of LSP1 in U251 and A172 cell lines were analyzed by qRT-PCR and western blotting. CCK8, colony formation and transwell assays were utilized to test glioblastoma cell malignant abilities. Furthermore, the associations between LSP1 and miR-920 were indentified by bioinformatics analysis and rescue assays. Moreover, the protein expression levels of p-JAK2, JAK2, p-STAT5 and STAT5, as the hallmark of JAK/STAT5 signaling, were detected by western blotting. The observations showed that LSP1 was highly augmented in glioblastoma samples. Additionally, up-regulation of LSP1 was associated with a unfavorable prognosis in glioblastoma patients. Biological experiments revealed that depletion of LSP1 significantly suppressed the proliferation, invasion and migration of U251 and A172 cells. MiR-920, as an upstream regulator of LSP1, negatively modulated LSP1 expression and promoted U251 cells malignant behaviors after miR-920 inhibitor treatment. However, together knockdown LSP1 and miR-920 inhibited these effects. Moreover, the expression levels of p-JAK2 and p-STAT5 were increased or decreased in U251 cells after transfection of miR-920 inhibitor or si-LPS1. Taken together, miR-920 might blocked the malignant development of glioblastoma cells, which is possibly realized by targeting LSP1 and modulation of JAK/STAT5 pathway. These findings implied that miR-920/LSP1 was a potential therapeutic target for glioblastoma treatment.

Effect of MIPU1 gene interference on biological behavior of U251 cell line.

To investigate effect of MIPU1 silence on proliferation, apoptosis, migration and invasion in U251 cells. The shRNA recombinant plasmids targeting MIPU1 gene was transfected into U251 cells. Western blotting was used to identify the inhibitory efficiency at 72 h after transfection. The cell viability was measured by MTT colorimetric assay. Hoechest staining and caspase-3 activity were used to detect apoptosis. Then wound healing assay and transwell migration assay were applied to detect the migration and invasion of cells. The expression of MIPU1 protein was effectively knocked down in transfected cells (P<0.05). The cellular proliferation was obviously inhibited and apoptosis was increased in shRNA-transfected MIPU1 cells (all P<0.05). The migration and invasion ability of cells transfected with positive plasmid was lower than that in the control group (P<0.05). Down-regulation of MIPU1 can promote apoptosis while inhibit the proliferation, invasion, and migration of U251 cells.

Effects of small nucleolar RNA SNORD44 on the proliferation, apoptosis and invasion of glioma cells.

To master the effect of small nucleolar RNA, SNORD44, on the proliferation, apoptosis and invasion of glioma cells and its relevant mechanism. SNORD44 and GAS5 expression in glioma tissues and cells was detected through qRT-PCR. Then, the glioma cell lines (U87 and U251) were divided into different groups with different treatments. Cell proliferation was determined by MTT assay, while the abilities of the cell migration and invasion were measured by wound-healing test and Transwell assay, respectively. Cell apoptosis were detected by flow cytometry and TUNEL assay. The expression of apoptosis proteins was quantified through Western blotting. Finally, the xenograft models were established on nude mice to investigate the effects of SNORD44 on the growth of glioma and the expressions of Ki67, MMP2 and MMP9 in vivo. SNORD44 and GAS5 were down-regulated in glioma tissues and cells in a positive correlation. Either SNORD44 or GAS5 overexpression decreased the proliferation, invasion and migration of U87 and U251 cells with the up-regulation of apoptosis rates, as well as the expressions of cleaved PARP, caspase 3, caspase 8 and caspase 9. Moreover, the in vivo experiment showed that overexpression of SNORD44 blocked the growth of glioma xenograft in nude mice accompanying with the inhibition of Ki67, MMP2 and MMP9 expressions. The combination overexpression of SNORD44 and GAS5 gained better inhibitory effects on glioma cells. Overexpression of SNORD44 and GAS5 activate the caspase-dependent apoptosis pathway to facilitate the apoptosis with the inhibited proliferation, invasion and migration of glioma cells.

PKA at a Cross-Road of Signaling Pathways Involved in the Regulation of Glioblastoma Migration and Invasion by the Neuropeptides VIP and PACAP.

Glioblastoma (GBM) remains an incurable disease, mainly due to the high migration and invasion potency of GBM cells inside the brain. PI3K/Akt, Sonic Hedgehog (SHH), and PKA pathways play major regulatory roles in the progression of GBM. The vasoactive intestinal peptide (VIP) family of neuropeptides and their receptors, referred in this article as the “VIP-receptor system”, has been reported to regulate proliferation, differentiation, and migration in a number of tumor cell types and more particularly in GBM cells. These neuropeptides are potent activators of the cAMP/PKA pathway. The present study aimed to investigate the cross-talks between the above cited signaling cascades. Regulation by VIP-related neuropeptides of GBM migration and invasion was evaluated ex vivo in rat brain slices explanted in culture. Effects of different combinations of VIP-related neuropeptides and of pharmacological and siRNA inhibitors of PKA, Akt, and of the SHH/GLI1 pathways were tested on GBM migration rat C6 and human U87 GBM cell lines using the wound-healing technique. Quantification of nuclear GLI1, phospho-Akt, and phospho-PTEN was assessed by western-immunoblotting. The VIP-receptor system agonists VIP and PACAP-38 significantly reduced C6 cells invasion in the rat brain parenchyma ex vivo, and C6 and U87 migration in vitro. A VIP-receptor system antagonist, VIP10-28 increased C6 cell invasion in the rat brain parenchyma ex vivo, and C6 and migration in vitro. These effects on cell migration were abolished by selective inhibitors of the PI3K/Akt and of the SHH pathways. Furthermore, VIP and PACAP-38 reduced the expression of nuclear GLI1 while VIP10-28 increased this expression. Selective inhibitors of Akt and PKA abolished VIP, PACAP-38, and VIP10-28 effects on nuclear GLI1 expression in C6 cells. PACAP-38 induced a time-dependent inhibition of phospho-Akt expression and an increased phosphorylation of PTEN in C6 cells. All together, these data indicate that triggering the VIP-receptor system reduces migration and invasion in GBM cells through a PKA-dependent blockade of the PI3K/Akt and of the SHH/GLI1 pathways. Therefore, the VIP-receptor system displays anti-oncogenic properties in GBM cells and PKA is a central core in this process.

FBW7 is associated with prognosis, inhibits malignancies and enhances temozolomide sensitivity in glioblastoma cells.

F‐box and WD repeat domain‐containing 7 (FBW7) is a SCF‐type E3 ubiquitin ligase targeting a multitude of oncoproteins for degradation. Acting as one of the most important tumor suppressors, it is frequently inactivated in various tumors. In this study we aimed to evaluate the relationship of FBW7 with glioma pathology and prognosis, and examine its effect in glioma malignancies and temozolomide (TMZ)‐based therapy. Clinical tissues and TCGA database analysis revealed that FBW7 expression was correlated inversely with glioma histology and positively with patient survival time. Lentivirus transfection‐induced FBW7 overexpression significantly suppressed proliferation, invasion and migration of U251 and U373 cells, whereas knockdown of FBW7 by targeted shRNA promoted proliferation, invasion and migration of glioma cells. Most importantly, the expression level of FBW7 was found to affect the 50% inhibitory concentration (IC50) of U251 and the TMZ‐resistant variant. Combining TMZ with FBW7 overexpression notably increased the cytotoxicity compared to TMZ treatment alone, which was conversely attenuated by FBW7 knockdown. Moreover, flow cytometry (FC) analysis showed overexpression of FBW7, TMZ or the combination‐increased proportion of G2/M arrest and the apoptotic rate, whereas FBW7 inhibition reduced G2/M arrest and apoptosis in U251 cells. Finally, mechanistic study found that FBW7 overexpression downregulated Aurora B, Mcl1 and Notch1 levels in a time‐dependent pattern and this expressional suppression was independent of TMZ. These findings collectively demonstrate the critical role of FBW7 as a prognostic factor and a potential target to overcome chemoresistance of glioblastoma.

Design, synthesis and biological evaluation of novel 5-hydroxy-2-methyl-4H-pyran-4-one derivatives as antiglioma agents.

d-2-Hydroxyglutarate (d-2HG) is frequently found in human brain cancers. Approximately 50-80% of grade II glioma patients have a high level of d-2HG production, which can lead to cancer initiation. In this study, a series of novel 5-hydroxy-2-methyl-4H-pyran-4-one derivatives were designed and synthesized as antiglioma agents, and their related structure-activity relationships are discussed. Among these novel compounds, 4a exhibited promising anti-proliferative activity against glioma HT1080 cells and U87 cells with an IC50 of 1.43 μM and 4.6 μM, respectively. Further studies found that the most active compound (4a) shows an 86.3% inhibitory rate against the intracellular production of d-2HG at 1 μM, and dramatic inhibitory effects, even at 1 μM on the colony formation and migration of U87 and HT1080 cells.

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.

Study on synergistic effect of Rac1 inhibitor and temozolomide in inhibiting proliferation and invasiveness of glioma cells in vitro

Objective To investigate whether Rac1 inhibitor has a synergistic effect on temozolomide (TMZ) in inhibiting the proliferation, migration and invasiveness of glioma cells. Methods Human glioma cell lines U87 and U251 were cultured by Rac1 inhibitor, TMZ or Rac1 inhibitor combined with TMZ. They were divided into 4 groups: control group, Rac1 inhibitor group, TMZ group and Rac1 inhibitor + TMZ group. The proliferation, migration and invasiveness of glioma cells were detected by methyl thiazolyl tetrazolium (MTT), migration assay and Transwell assay. Results After cultured by Rac1 inhibitor, TMZ or Rac1 inhibitor + TMZ, the cell proliferation of U87 and U251 were inhibited ( P < 0.05, for all). The inhibiting effect of TMZ was stronger than Rac1 inhibitor ( P < 0.05, for all), while Rac1 inhibitor + TMZ was much stronger ( P < 0.05, for all). U87 and U251 migration [cells/low power field (LPF)] in Rac1 inhibitor group (39.00 ± 9.53, 37.00 ± 5.56), TMZ group (42.00 ± 8.54, 36.00 ± 9.00) and Rac1 inhibitor + TMZ group (18.67 ± 10.54, 14.33 ± 5.00) was lower than that in control group (78.00 ± 11.53, 75.00 ± 4.00), and the differences were significant (U87: P = 0.001, 0.001, 0.000; U251: P = 0.000, for all). U87 and U251 cell invasiveness (cells/LPF) in Rac1 inhibitor group (30.33 ± 3.51, 40.67 ± 4.04), TMZ group (24.00 ± 2.64, 37.33 ± 4.51) and Rac1 inhibtor + TMZ group (11.00 ± 2.00, 15.33 ± 2.52) was lower than that in control group (64.33 ± 4.04, 77.33 ± 3.06), and the differences were significant (U87: P = 0.000, for all; U251: P = 0.000, for all). In paired comparison among different groups, the migration (U87: P = 0.021, 0.011; U251: P = 0.002, 0.003) and invasiveness (U87: P = 0.000, 0.001; U251: P = 0.000, 0.000) of cells in Rac1 inhibitor + TMZ group were significantly decreased than those in other groups. Conclusions Both Rac1 inhibitor and temozolomide can inhibit the proliferation, migration and invasiveness of glioma cells, while Rac1 inhibitor has a significantly synergistic effect on temozolomide. DOI: 10.3969/j.issn.1672-6731.2016.08.009

Correlation of C-X-C chemokine receptor 2 upregulation with poor prognosis and recurrence in human glioma.

C-X-C chemokine receptor 2 (CXCR2), a member of the G-protein-coupled receptor family, is an interleukin-8 receptor and results in the activation of neutrophils. To date, CXCR2 has been identified with many cell events, including inflammation, neovascularization, metastasis, and cell carcinogenesis. This study aimed to investigate alterations in the expression of CXCR2 in patients with brain gliomas and relationships with pathological grades and clinicopathological characteristics. Brain tissue specimens from 60 patients with glioma and 15 patients undergoing surgery for epilepsy (controls) were detected using streptavidin-perosidase immunohistochemistry. Western blotting was used to evaluate CXCR2 protein levels with fresh tissues derived from glioma cases or controls. Correlations between CXCR2 expression and clinicopathological characteristics were analyzed using SPSS software. The results showed high-grade gliomas with high CXCR2 expression as compared with normal tissues. The expression of CXCR2 was significantly related to high grades and recurrence of tumor but not to age or sex. During an in vitro wound healing assay, U251 migration was reduced when the CXCR2-specific inhibitor SB225002 was applied. Our results suggested that the high expression of CXCR2 in gliomas was closely correlated to the degree of malignancy and recurrence and that CXCR2 inhibition decreased the migration of glioma cells. Therefore, CXCR2 may serve as a potential therapeutic target for the recurrence and migration of gliomas.

HMGCR positively regulated the growth and migration of glioblastoma cells

The metabolic program of cancer cells is significant different from the normal cells, which makes it possible to develop novel strategies targeting cancer cells. Mevalonate pathway and its rate-limiting enzyme HMG-CoA reductase (HMGCR) have shown important roles in the progression of several cancer types. However, their roles in glioblastoma cells remain unknown. In this study, up-regulation of HMGCR in the clinical glioblastoma samples was observed. Forced expression of HMGCR promoted the growth and migration of U251 and U373 cells, while knocking down the expression of HMGCR inhibited the growth, migration and metastasis of glioblastoma cells. Molecular mechanism studies revealed that HMGCR positively regulated the expression of TAZ, an important mediator of Hippo pathway, and the downstream target gene connective tissue growth factor (CTGF), suggesting HMGCR might activate Hippo pathway in glioblastoma cells. Taken together, our study demonstrated the oncogenic roles of HMGCR in glioblastoma cells and HMGCR might be a promising therapeutic target.

Abstract C294: Involvement of iNOS/NO and ROS in a reciprocal interaction of glioma and microglia cells via TNF-α and MCP-1 production.

Abstract The tumor microenvironment plays a critical role in glioblastoma invasion and progression, and microglia is one of the most abundant immune cells in the tumor. Using transwell co-culture methods with glioma cells (U87 and C6) and microglia cells BV-2, increased migration of glioma cells U87 and C6 by microglia BV2 cells or NO donor sodium nitro prusside (SNP) accompanied by increased NO production was observed, and BV2 co-culture-induced migration of U87 and C6 was blocked by adding a nitric oxide synthase (NOS) inhibitor N-nitro L-arginine methyl ester (NAME). The conditioned mediums (CM) derived from both glioma cells U87 and C6 (U87-CM and C6-CM) were prepared, CMs from glioma cells significantly induced iNOS protein and NO production cells in according with an occurrence of activated macrophages and without alternations in cell viability and cell cycle progression in microglia BV2. ERK inhibitor U0126 and JNK inhibitor SP600125 suppressed U87-CM and C6-CM-induced iNOS/NO production with respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expression stimulated by U87-CM and C6-CM. A significant increase in intracellular peroxide by U87-CM and C6-CM was detected via DCHF-DA assay, and vitamin C (Vit C) and N-acetyl cysteine (NAC)-reduced intracellular peroxide levels with inhibition of iNOS/NO production and pERK/pJNK protein stimulated by U87-CM and C6-CM were identified in BV-2 cells. Contribution of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in the transwell co-culture system of glioma U87 and C6 with microglia BV-2 cells. Furthermore, TNF-α, MCP-1, CSF-1, and TGF-β were applied, and TNF-α and MCP-1 induced iNOS protein expression in time and concentration-dependent manners, and expression of TNF-α and MCP-1 mRNA in U87 and C6 cells were detected by RT-PCR. Neutralization of TNF-α or MCP-1 in U87-CM and C6-CM using TNF-α or MCP-1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF-α or MCP-1 was identified in BV2 cells. The reciprocal activation of glioma cells and microglia via ROS-dependent iNOS/NO elevation at least in part mediated by TNF-α and MCP-1 is elucidated. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C294. Citation Format: Yen-chou Chen. Involvement of iNOS/NO and ROS in a reciprocal interaction of glioma and microglia cells via TNF-α and MCP-1 production. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C294.