Proliferation Of Glioma Cell Lines Research Articles

The role of ST3GAL4 in glioma malignancy, macrophage infiltration, and prognostic outcomes

BackgroundGlioma, a prevalent malignancy of the brain and spinal cord, poses a considerable threat to human health. The association between aberrant sialic acid modification and glioma progression has been suggested, but the precise mechanism is still elusive. ST3GAL4, a sialoglycosyltransferase, is implicated in increased metastatic potential and poor prognosis in various cancers; however, its specific role in glioma requires further elucidation. MethodsWe evaluated ST3GAL4 expression levels and their clinical relevance using the TCGA database, and we assessed immune infiltration via the Tumor Immune Evaluation Resource (TIMER) database. In vitro experiments were performed to determine the effects of ST3GAL4 knockdown on glioma cell malignancy, with additional co-culture assays to assess its impact on macrophage phenotype. ResultsST3GAL4 expression was markedly elevated in glioma tissues compared to normal brain tissues, with a strong correlation to glioma patient clinical characteristics. Survival analyses and receiver operating characteristic (ROC) curves suggested that ST3GAL4 is a feasible diagnostic and prognostic biomarker for glioma. Knockdown studies revealed that ST3GAL4 inhibition reduces glioma cell line proliferation, migration, and invasion, while causing G1 phase cell cycle arrest. ST3GAL4 appears to mediate glioma progression through extracellular matrix reorganization and EMT signaling pathway activation, further contributing to M2 macrophage polarization and infiltration within the tumor microenvironment. ConclusionOur research highlights the critical role of ST3GAL4 in glioma development, positioning it as a promising candidate for diagnostic and therapeutic interventions.

PSMC2 promotes glioma progression by regulating immune microenvironment and PI3K/AKT/mTOR pathway

BackgroundGlioma, the most frequent and malignant central nervous system (CNS) cancer, has a bad outcome. Proteasome 26S subunit ATPase 2 (PSMC2) is an essential part of the 26S proteasome and promotes the development of several tumors. However, the pathway and function of PSMC2 in glioma have not been unelucidated. MethodsThis study analyzed PSMC2 expression in glioma tissues and its predictive significance for patients. We examined the link between PSMC2 and DNA methylation, immune cell infiltration, tumor immune cycle, immune cell homeostasis, and immune checkpoints. Subsequently, immunohistochemistry and in vitro trials were employed to validate the expression, prognostic potential, and function of PSMC2 in glioma. The mechanisms of PSMC2 in glioma were further explored. ResultsOur study revealed that PSMC2 expression increased in glioma tissues contrasted with healthy tissues, and patients with high PSMC2 glioma exhibited poor overall survival (OS) compared to the low-PSMC2 group. Immune profile analysis revealed that PSMC2 was positively related to immunosuppressive cell infiltration and immune checkpoints and adversely related to the cancer immune cycle and immune cell homeostasis. In cell-based investigations, the inhibition of PSMC2 was found to effectively suppress the aggressiveness and proliferation of glioma cell lines while also enhancing cell cycle arrest and promoting cell death. Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA), and in vitro experiments showed that PSMC2 promoted glioma development through the PI3K/AKT/mTOR pathway. ConclusionsPSMC2 was upregulated in glioma and promoted cancer progression by modulating the tumor immune microenvironment, cancer cell biological behavior, immune cell homeostasis, and the PI3K/AKT/mTOR pathway, providing a new option to treat glioma.

EXTH-55. ALL-IN-ONE GENETIC CONSTRUCT TO KNOCKDOWN MIR-21 REDUCES GLIOMA PROLIFERATION IN VITRO

Abstract Glioma, the most aggressive tumor of the CNS has poor patient outcome with limited effective treatments. CRISPR-CAS technology recently opened a new avenue for gene therapy and has been previously used to target non-coding RNAs, including microRNA-21, an oncogene associated with glioma progression. Here we show that an all-in-one construct containing both Staphylococcus Aureus Cas9 (SaCas9) guided by a single-guide RNA (sgRNA) specifically targeting miR-21 resulted in a deletion causing a downregulation of miR-21 expression levels and reduced proliferation in glioma cell lines. Here, we aim to design a single adeno-associated vector (rAAV) vector platform that express both saCas9 and sgRNA to target miR-21 in a glioma syngeneic mouse model. With the future goal to engineer a single AAV that can achieve therapeutic genome editing outcomes.

Kaempferol and Biomodified Kaempferol from Sophora japonica Extract as Potential Sources of Anti-Cancer Polyphenolics against High Grade Glioma Cell Lines.

The enzymatic hydrolysis of the extract of Sophora japonica by two glycosyl hydrolases (hesperidinase and galactosidase) was performed in order to obtain kaempferol (KPF)-enriched extract with an enhanced anticancer activity. The current study examined the effectiveness of both Sophora japonica extracts (before (KPF-BBR) and after (KPF-ABR) bioconversion reactions) in reducing cell viability and inducing apoptosis in human high-degree gliomas in vitro. Cytotoxicity was determined using an MTT assay. The effects of both compounds on the proliferation of glioma cell lines were measured using trypan blue exclusion, flow cytometry for cell cycle, wound healing (WH), and neurosphere formation assays. Cellular apoptosis was detected by DNA fragmentation and phosphatidylserine exposure. qPCR and luciferase assays evaluated NF-kB pathway inhibition. The survival rate of NG-97 and U-251 cells significantly decreased in a time- and dose-dependent manner after the addition of KPF-BBR or KPF-ABR. Thus, a 50% reduction was observed in NG-97 cells at 800 µM (KPF-BBR) and 600 µM (KPF-ABR) after 72 h. Both compounds presented an IC50 of 1800 µM for U251 after 72 h. The above IC50 values were used in all of the following analyses. Neither of the KPF presented significant inhibitory effects on the non-tumoral cells (HDFa). However, after 24 h, both extracts (KPF-BBR and KPF-ABR) significantly inhibited the migration and proliferation of NG-97 and U-251 cells. In addition, MMP-9 was downregulated in glioma cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus KPF-BBR and TPA+KPF-ABR compared with the TPA-treated cells. Both KPF-BBR and KPF-ABR significantly inhibited the proliferation of glioma stem cells (neurospheres) after 24 h. DNA fragmentation assays demonstrated that the apoptotic ratio of KPF-ABR-treated cell lines was significantly higher than in the control groups, especially NG-97, which is not TMZ resistant. In fact, the flow cytometric analysis indicated that KPF-BBR and KPF-ABR induced significant apoptosis in both glioma cells. In addition, both KPF induced S and G2/M cell cycle arrest in the U251 cells. The qPCR and luciferase assays showed that both KPFs downregulated TRAF6, IRAK2, IL-1β, and TNF-α, indicating an inhibitory effect on the NF-kB pathway. Our findings suggest that both KPF-BBR and KPF-ABR can confer anti-tumoral effects on human cell glioma cells by inhibiting proliferation and inducing apoptosis, which is related to the NF-κB-mediated pathway. The KPF-enriched extract (KPF-ABR) showed an increased inhibitory effect on the cell migration and invasion, characterizing it as the best antitumor candidate.

Expression and potential role of FOSB in glioma.

BackgroundFOSB is reported to be an oncogene in a variety of tumors. However, the expression and role of FOSB in glioma remain obscure. In this study, we aimed to explore the expression of FOSB in glioma and its biological role in glioblastoma multiforme (GBM).MethodsWestern blot, immunohistochemical staining, and quantitative real-time polymerase chain reaction (RT-qPCR) were used to detect the expression of FOSB in clinical samples. FOSB was knocked down in cells to determine the effects of FOSB on the phenotypic changes of tumors by plate cloning, CCK-8 assay, and Transwell assay. Finally, subcutaneous tumorigenesis in nude mice was used to observe the tumorigenesis of glioma cell lines after the knockdown of the FOSB gene.ResultsFOSB expression was higher in glioma compared with normal brain tissue. After the downregulation of FOSB, the expression of cleaved caspase-3 increased. Plate cloning and CCK-8 experiments showed that the proliferation of glioma cell lines decreased. The Transwell assay demonstrated that the glioblastoma cell lines had lower migration ability after the knockdown of FOSB. Finally, the tumor volume of U87 glioma cells in group sh-FOSB was smaller than that in the control group. The TUNEL staining in vitro showed that the apoptosis of sh-FOSB glioma cells increased.ConclusionFOSB was highly expressed in glioma tissues. The viability of glioma cells decreased, and the ability of glioma cells to proliferate and migrate was reduced when FOSB was downregulated. Hence, FOSB may promote the development and migration of gliomas.

Inhibitory Mechanism of Combined Hydroxychavicol With Epigallocatechin-3-Gallate Against Glioma Cancer Cell Lines: A Transcriptomic Analysis.

Emerging reports have shown therapeutic potential of hydroxychavicol (HC) and epigallocatechin-3-gallate (EGCG) against cancer cells, however high concentrations are required to achieve the anticancer activity. We reported the synergy of low combination doses of EGCG+HC in glioma cell lines 1321N1, SW1783, and LN18 by assessing the effects of EGCG+HC through functional assays. Using high throughput RNA sequencing, the molecular mechanisms of EGCG+HC against glioma cell lines were revealed. EGCG/HC alone inhibited the proliferation of glioma cell lines, with IC50 values ranging from 82 to 302 µg/ml and 75 to 119 µg/ml, respectively. Sub-effective concentrations of combined EGCG+HC enhanced the suppression of glioma cell growth, with SW1783 showing strong synergism with a combination index (CI) of 0.55 and LN18 showing a CI of 0.51. A moderate synergistic interaction of EGCG+HC was detected in 1321N1 cells, with a CI value of 0.88. Exposure of 1321N1, SW1783, and LN18 cells to EGCG+HC for 24 h induces cell death, with caspase-3 activation rates of 52%, 57%, and 9.4%, respectively. However, the dose for SW1783 is cytotoxic to normal cells, thus this dose was excluded from other tests. EGCG+HC induced cell cycle arrest at S phase and reduced 1321N1 and LN18 cell migration and invasion. Combined EGCG+HC amplified its anticancer effect by downregulating the axon guidance process and metabolic pathways, while simultaneously interfering with endoplasmic reticulum unfolded protein response pathway. Furthermore, EGCG+HC exerted its apoptotic effect through the alteration of mitochondrial genes such as MT-CO3 and MT-RNR2 in 1321N1 and LN18 cells respectively. EGCG+HC dynamically altered DYNLL1 alternative splicing expression in 1321N1 and DLD splicing expression in LN18 cell lines. Our work indicated the pleiotropic effects of EGCG+HC treatment, as well as particular target genes that might be investigated for future glioma cancer therapeutic development.

LncRNA HAND2-AS1 attenuates glioma cell proliferation, invasion and migration by targeting CDK6

ABSTRACT Objective Long non-coding RNA (lncRNA) HAND2-AS1 has been indicated to play biological roles in several cancers. However, whether HAND2-AS1 could exert its role in glioma remains unknown. We aimed to investigate the function of HAND2-AS1 in the proliferation, invasion and migration and to explore its underlying molecular mechanism in glioma cells. Methods Reverse transcription-quantitative polymerase chain reaction analysis was conducted to determine the expression of HAND2-AS1 and cyclin-dependent kinases 6 (CDK6) in tissues or cells. Western blot analysis was used to detect the CDK6 protein expression. CCK-8 assay was adopted to determine the proliferation in glioma cell lines. Transwell assay was taken to evaluate the invasion and migration in glioma cell lines. Results Our results revealed that HAND2-AS1 expression was significantly downregulated in the glioma tissues and cell lines. Moreover, overexpression of HAND2-AS1 attenuated the proliferation, invasion, and migration in glioma cell lines. However, overexpression of CDK6 could partially block the inhibitory role of HAND2-AS1 on cell proliferation as well as cell invasion and migration in glioma cell lines. Conclusion LncRNA HAND2-AS1 may play a critical anti-tumorigenic role in glioma by negatively regulating CDK6 expression.

DDRE-25. INVESTIGATING MITOCHONDRIAL SLC25A TRANSPORTERS INVOLVED IN SUPPORTING BRAIN TUMOUR METABOLISM AND SURVIVAL UNDER HYPOXIC CONDITIONS

Advancements in prevention, detection and treatment over the last 40 years have significantly transformed cancer healthcare however there are a few cancers, such as brain tumours, which are consistently lagging behind. The most common adult brain tumour is glioma; a highly aggressive cancer that invades deep into the surrounding brain consequently making treatment challenging. The severe hypoxic nature of glioma adds further complications to therapeutic efficacy as hypoxia limits efficient drug delivery as well as increasing treatment resistance. Therapies that therefore target both the hypoxic tumour microenvironment and metabolic pathways that sustain growth have significant potential to improve patient prognosis. It is well known that cancer cells demonstrate an abnormal metabolism, resulting in an altered requirement for amino acids to aid uncontrolled proliferation. Furthermore, tumour metabolism can also be influenced by this hostile hypoxic microenvironment, leading to a more malignant phenotype. We are therefore interested in a family of mitochondrial transporters, SLC25A, which translocate numerous solutes across the mitochondrial membrane and are crucial for many metabolic reactions. TCGA analysis has shown that many of these amino acid carriers are upregulated in glioma. Remarkably however, around 23 of the 53 mammalian SLC25A members lack defined substrate selectivity and so we are interested in identifying which transporters are particularly important in the metabolic adaptation to hypoxia. Using CRISPR and siRNA technologies we have identified transporters that are functionally required to maintain cell proliferation of glioma cell lines and patient tumour cells. Furthermore, using stable isotope-enriched nutrients, we have identified novel means by which glioma cell metabolism can be perturbed by inhibition of these transporters. Characterising which SLC25A transporters are important for hypoxic tumour metabolism could therefore expose a way to exploit these hypoxic areas subsequently making them more vulnerable to treatment and thus impacting patient survival.

Gomisin J inhibits the glioma progression by inducing apoptosis and reducing HKII-regulated glycolysis

Glioma is a leading cause of central nervous system malignant tumor-associated deaths in the world. However, the molecular mechanisms for glioma progression are still unclear, lacking effective therapeutic strategies. Gomisin J (GomJ) is a derivative of lignan compound, and shows regulatory effects on virus, oxidative stress and tumor progression. However, the role of GomJ in the meditation of glioma progression has not been explored. In this study, we found that GomJ markedly reduced the proliferation of glioma cell lines. Mitochondrial apoptosis was highly induced by GomJ, as evidenced by the significantly up-regulated expression of cytoplastic Cyto-c and cleaved Caspase-3. In addition, mitochondrial membrane potential (MMP) and oxidative stress were highly triggered in GomJ-incubated glioma cells, accompanied with the glycolysis suppression. Importantly, we found that GomJ could dramatically reduce the expression of hexokinase II (HKII) in glioma cells. At the same time, the dissociation of HKII from voltage-dependent anion channel (VDAC) in mitochondria was markedly induced by GomJ, contributing to glycolytic repression. The in vivo experiments confirmed that GomJ obviously reduced the growth of glioma with HKII reduction and few side effects. Taken together, these results demonstrated that GomJ could inhibit the proliferation, induce apoptosis and restrain HKII-regulated glycolysis during glioma progression. Herein, GomJ with few toxicity might be served as a potential therapeutic strategy for the treatment of glioma in humans.

Polyphyllin VI Induces Apoptosis and Autophagy via Reactive Oxygen Species Mediated JNK and P38 Activation in Glioma.

BackgroundPolyphyllin VI (PPVI), a bioactive component derived from a traditional Chinese herb Paris polyphylla, exhibits potential antitumor activity against hepatocellular carcinoma, as well as breast and lung cancers. However, its effect on glioma remains unknown.MethodsFive glioma cell lines (U251, U343, LN229, U87 and HEB) and an animal model were employed in the study. Anti-proliferation effects of PPVI were first determined using CCK-8 cell proliferation and clone formation assays, then reactive oxygen species (ROS), cell cycle progression and apoptosis effects measured by flow cytometry. The effect of PPVI on protein expression was quantified by Western blot analysis.ResultsData showed that PPVI inhibited the proliferation of glioma cell lines by modulating the G2/M phase. Additionally, incubation of cells with PPVI promoted apoptosis, autophagy, increased accumulation of ROS and activated ROS-modulated JNK and p38 pathways. On the other hand, N-acetyl cysteine, a ROS inhibitor, attenuated PPVI-triggered effects. Furthermore, JNK and p38 inhibitors ameliorated PPVI-triggered autophagy and apoptosis in glioma cells. In vivo assays showed that PPVI inhibited tumor growth of U87 cell line in nude mice.ConclusionOverall, these data suggested that PPVI might be an effective therapeutic agent for glioma.

DDIS-05. IDENTIFICATION OF 2-FLUORO PALMITIC ACID AS A POTENTIAL THERAPEUTIC AGENT AGAINST GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) represents the most aggressive and frequent type of primary malignant brain tumors with a dismal clinical outcome. Though adjuvant temozolomide (TMZ) chemotherapy followed by surgical resection can extend patient`s post-operative survival, a considerable number of GBM cases are refractory to TMZ. Novel therapeutic agents that can overcome chemoresistance against TMZ are urgent. Drug repositioning is a process of identifying new indications for existing drugs and provides potential possibilities to discover new drugs. In this study, we explored novel anti-glioma agents which enhances the effect of TMZ with the strategy of drug repositioning. METHODS Drug library which contains 1300 diverse chemical compounds was screened using 2 kinds of glioma stem cell (GSC) lines to select novel therapeutic candidate. The effect of candidate drug on the proliferation of GSCs was estimated by sphere formation assay. To evaluate its efficacy against glioma cell biology, proliferation assay, matrigel invasion assay were performed. To clarify the mechanism of drug effects, we investigated target molecules by gelatin zymography and western blot. RESULTS Acyl CoA synthetase inhibitor named 2-fluoropalmitic acid (2-FPA) was selected as a novel candidate. 2-FPA suppressed proliferation of glioma cell lines by single administration and/or combination with TMZ. The sphere formation of GSCs was suppressed by combination therapy. Combination therapy enhanced TMZ effect. 2-FPA also suppressed invasion of glioma cell lines in a dose dependent manner. 2-FPA suppressed MMP-2 activity and phosphorylation of STAT3. CONCLUSION 2-FPA was identified as a novel potential therapeutic agent against GBM.

Silencing Of MAGI1 Promotes The Proliferation And Inhibits Apoptosis Of Glioma Cells Via The Wnt/β-Catenin And PTEN/AKT Signaling Pathways.

BackgroundMembrane-associated guanylate kinase (MAGUK) with inverted orientation protein 1 (MAGI1) is a novel member of the MAGUK family with a vital role in tumor progression related to invasion and metastasis. However, the function of MAGI1 in glioma is currently unknown. We therefore analyzed the expression of MAGI1 protein in human glioma samples, glioma cell lines and glioma stem cells (GSCs), and explored its effects on glioma cell proliferation and apoptosis.MethodsMAGI1 expression in glioma tissues was examined by Western blotting and real-time polymerase chain reaction and its relationships with clinical pathological features were analyzed. The effects of MAGI1 knockdown on the proliferation of glioma cell lines and GSCs were detected by CCK8 and colony-formation assays, and apoptosis was assessed by flow cytometry. We also investigated the effects of MAGI1 silencing on protein expression levels of epithelial-mesenchymal transition biomarkers, as well as β-catenin, cyclin D1, PTEN and phospho-Akt by Western blotting.ResultsMAGI1 was significantly downregulated in glioma tissues and its expression was related to cancer progression. Silencing of MAGI1 in both glioma cell lines and GSCs enhanced proliferation and inhibited apoptosis. MAGI1 knockdown also significantly increased the expression levels of N-cadherin, vimentin, β-catenin, cyclin D1 and phospho-Akt and reduced the expression of E-cadherin and PTEN.ConclusionsOur results indicated that MAGI1 might play a vital role in glioma progression and may represent a potential therapeutic target for the treatment of glioma.

The effect of circ-ZEB1 on the biological characteristics of glioma cells

Objective To investigate the effects of circ-ZEB1 on proliferation, invasion and migration of glioma cells. Methods Real-time quantitative PCR (polymerase chain reaction) was used to detect the expression of circ-ZEB1 in 30 glioma tissues and 8 normal brain tissues(obtained from Department of Neurosurgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University), glioma cell lines (A172 and U251) and normal glial cells (HA1800). A172 and U251 cells were transfected with si-circ-ZEB1 to knock down circ-ZEB1. According to the type of si-circ-ZEB1, it was divided into si-1 group and si-2 group (transfected with si-circ-ZEB1-1, si-circ-ZEB1-2, respectively), and a control group was established (siRNA-NC as negative groups transfected with siRNA-NC). The effect of circ-ZEB1 on the proliferation of glioma cell lines was detected by CCK-8 assay. Scratch assay and Transwell assay were used to observe the effects of circ-ZEB1 on the migration and invasion in glioma cells respectively. CircNet database was used to examine and evaluate of circRNA-miRNA gene regulatory network of circ-ZEB1. The dual luciferase reporter assay was used to validate the interaction of circ-ZEB1/miR-140-3p in the gene regulatory network of circRNA-miRNA. Results The expression of circ-ZEB1 was higher in glioma tissues than in normal tissues (12.16±1.31 vs. 6.84±0.56, P=0.001). Circ-ZEB1 was over-expressed in high-grade glioma tissues compared with low-grade gliomas (13.77±3.27 vs. 7.50±1.82, P=0.001). The expression levels of circ-ZEB1 in A172 and U251 were 2.91±0.30 and 3.32±0.20, respectively, compared with HA1800 (both P<0.01). The results of CCK-8 showed that the growth of circ-ZEB1 knockdown group (si-1 group and si-2 group) was significantly inhibited compared with the control group. The proliferation abilities of A172 cells were decreased by 40.0% and 57.1%, respectively (both P<0.01) and those of U251 cells were decreased by 35.3% and 48.5%, respectively (both P<0.01). In the scratch test, the migration of circ-ZEB1 knockdown group was significantly inhibited (both P<0.05). Transwell assay showed that the invasive abilities of glioma cells in si-1 group and si-2 group were lower than those of the control group (both P<0.01). The gene regulatory networks of circRNA-miRNAs showed that circ-ZEB1 interacted with a variety of miRNAs, which affected the regulation of multiple miRNAs and their downstream proteins. The dual luciferase reporter assay also confirmed the interaction of circ-ZEB1/miR-140-3p in the gene regulatory network of circRNA-miRNA. Conclusions Circ-ZEB1 is over-expressed in glioma tissues and cell lines. Down-regulation of circ-ZEB1 could decrease cell proliferation and cell migration in gliomas. The gene regulatory network of circRNA-miRNA suggests that circ-ZEB1 functions by interacting with a variety of miRNAs. Key words: Glioma; Circ-ZEB1; Cell proliferation; Cell migration assays

Glioma cell proliferation is inhibited by miR-342-3p, miR-377 / E2F1 signaling pathway.

Recent years, micoRNAs (miRNAs) have been reported to be critical regulators to influence tumor genesis or further progression by directly targeting downstream tumor related genes in glioma. However, there're still many underlying mechanisms related to miRNAs signaling pathway remain to be uncovered in glioma. In the present study, we found that miR-342-3p and miR-377 inhibited the glioma cell line proliferation and arrested the cell cycle at G1 phase. Inhibition of the function of miR-342-3p and miR-377 promoted the cell proliferation. miR-342-3p and miR-377 target the E2F1 3'UTR to repress its expression on both mRNA and protein level. Downregulation of E2F1 inhibited the cell proliferation and arrested the cell cycle. Overexpression of E2F1 blocked the proliferation repression caused by miR-342-3p or miR-377 in glioma cells. This study showed the function of miR-342-3p, miR-377/E2F1 axis in regulating glioma cells proliferation and provided the potential therapeutic target.

PiRNA-8041 is downregulated in human glioblastoma and suppresses tumor growth in vitro and in vivo.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that partner with PIWI proteins to protect germline tissues from destabilizing transposon activity. While the aberrant expression of PIWI proteins has been linked with poor outcomes for many cancers, less is known about the expression or function of piRNAs in cancer. We performed array-based piRNA expression profiling in seven pairs of normal brain and glioblastoma multiforme (GBM) tissue specimens, and identified expression of ~350 piRNAs in both tissues and a subset with dysregulated expression in GBM. Over-expression of the most down-regulated piRNA in GBM tissue, piR-8041, was found to reduce glioma cell line proliferation, induce cell cycle arrest and apoptosis, and inhibit cell survival pathways. Furthermore, pre-treatment with piR-8041 significantly reduced the volume of intracranial mouse xenograft tumors. Taken together, our study reveals reduced expression in GBM of piR-8041 and other piRNAs with tumor suppressive properties, and suggests that restoration of such piRNAs may be a potential strategy for GBM therapy.

Downregulation of RNF138 inhibits cellular proliferation, migration, invasion and EMT in glioma cells via suppression of the Erk signaling pathway.

Glioma is the most common adult malignant primary brain tumor; however, the effect of chemotherapy is often limited by drug-resistance and poor prognosis is common. Ring finger protein 138 (RNF138) belongs to the E3 ligase family, and has significantly higher expression level in glioma tissue than in noncancerous brain tissues. Epithelial-mesenchymal-transition (EMT) has a critical role in cancer invasion and metastasis, ultimately leading to increased cell motility and resistance to genotoxic agents. Extracellular-signal regulated kinase (Erk) pathways promote the growth of glioma cells and enhance tumor invasion, with a role in the progression of EMT. However, the association between RNF138 and human glioma progression remains poorly understood. Relatively little is known about the association between RNF138, Erk, and EMT in glioma progression. In the current study, experiments were performed to explore the potential roles and mechanisms of RNF138 in glioblastoma in vitro and in vivo. Glioma cell line proliferation, migration and invasion were inhibited by knockdown of RNF138 in vitro. By lowering the RNF138 expression, cleaved caspase3 and E-cadherin were upregulated, while phospho-Erk1/2, vimentin, MMP2, HIF-1α and VEGF were downregulated in U87 and U251 cells in vitro. In vivo findings revealed that the growth of U87 cell-transplanted tumors in nude mice was inhibited in tumors with RNF138 knockdown. These findings suggested that downregulation of RNF138 inhibited glioma cell proliferation, migration, and invasion, and reversed EMT, potentially via Erk signaling pathway. Therefore, RNF138 may be a potential therapeutic target against glioma.

Antitumor effect of levetiracetam combined with temozolomide in glioma cell lines.

e13530 Background: The antitumor effect oftemozolomide (TMZ) is still remitted for portion of glioma patients, and if anti-epilepcy drug levetiracetam (LEV) may help TMZ for anti-glioma is not clear. Methods: The cell proliferation with Cell Counting Kit-8 (CCK-8) assay was performed in two O6-methylguanine DNA methyltransferase (MGMT) positive cell lines (T98G and U138) and two other MGMT negative cell lines (SKMG-4 and U87). The cells were treated with LEV and TMZ alone or in combination. Results: LEV inhibited the proliferation of glioma cell lines but not reach therapeutic effect, especially in U87cell line. The 50% inhibitory concentration (IC50) of TMZ was significantly decreased in cell lines (T98G, U138 and SKMG-4, P&lt; 0.05) which were treated with TMZ and LEV, but no significantly changed in U87 (P&gt; 0.05). Conclusions: LEV may inhibit the proliferation of glioma cell lines (both MGMT- positive and MGMT-negative) and also sensitizes cell lines (T98G, U138 and SKMG-4, but not U87) to TMZ.

EXTH-14. ADD ON EFFECT OF EXISTING DRUGS TO TMZ FOR GLIOMA TREATMENT

Temozolomide has been currently used as first line chemotherapy agent for GBM, and the best 5-year survival rate is only 9.8%. For searching methods to enhance antitumor efficacy of TMZ, several agents that are not antitumor medications have been tested. Interferons (IFNs), an immonomodulatory agent, are capable of sensitizing GBM to TMZ via multiple mechanism. Our in vitro and in vivo study has revealed that IFNα/β can enhance the cytotoxicity of TMZ, which are correlated with the down regulation of NF- κB and MGMT expression. Recent studies suggested that tumor cytotoxicity and radiosensitivity associated with histone deacetylases (HDACs). VPA (2-propylpentanoic acid) is one of the most interesting HDAC inhibitors. Recently, findings that VPA can induce hyperacetylation of histones means it is a possible agent that can enhance tumor cytotoxicity and radiosensitivity. We have found that VPA possess mild antitumor activity, may induce apoptosis (mild) as well as autophagy in glioma cell lines, and can increase antitumor activity of TMZ which could be related to enhancing autophagy. We investigated the anti-glioma efficacy of dihydroartemisinin (DHA) and combined with temozolomide (TMZ) on human glioma cell lines in vitro and in vivo. Our data suggest that DHA can inhibit proliferation of glioma cell lines and increase antitumor efficacy of TMZ, and possibly through inducing autophagy. We have tested and found that Beta-elemene (β-ELE) could compensate for TMZ to kill both glioma stem-like cells (GSLCs) and non-stem like cancer cells. Notch1 might be a downstream target of β-ELE. In conclusion, repurposing existing drugs which are not antitumor agents could be one of the permission ways for glioma treatment.

Methylation Status of the RIZ1 Gene Promoter in Human Glioma Tissues and Cell Lines.

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1), a strong tumor suppressor, is silenced in many human cancers. Our previous studies showed that RIZ1 expression was negatively correlated with the grade of glioma and was a key predictor of patient survival. Therefore, RIZ1 could be a potential tumor suppressor during glioma pathogenesis, although the mechanism underlying RIZ1 gene inactivation in gliomas is unknown. We investigated the methylation status of the RIZ1 promoter in human glioma tissues and four glioblastoma (GBM) cell lines, and verified the effect of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-CdR) on RIZ1 transcription and cell proliferation. Methylation-specific PCR (MSP) was performed to determine RIZ1 promoter methylation in human glioma specimens. The correlation between RIZ1 hypermethylation in tumors and clinicopathological features also was analyzed. 5-Aza-CdR treatment was used to reactivate gene expression silenced by hypermethylation in the U87 glioblastoma cell line, and real-time PCR was then used to measure RIZ1 expression. The ability of 5-aza-CdR to inhibit the proliferation of glioma cell lines whose RIZ1 promoters were hypermethylated was measured by bromodeoxyuridine (BrdU) incorporation. Among 51 human glioma specimens, RIZ1 promoter methylation was detected in 23 cases. Clinicopathological evaluation suggested that RIZ1 hypermethylation was negatively associated with tumor grade and patient age (P<0.05). Hypermethylation of the RIZ1 promoter was detected in the U87 and U251 cell lines. RIZ1 mRNA expression in U87 cells was upregulated after treatment with 5-aza-Cdr, which correlated with inhibition of cell proliferation in a time- and concentration-dependent manner. Promoter hypermethylation may play an important role in the epigenetic silencing of RIZ1 expression in human glioma tissues and GBM cell lines.

Overexpression of transient receptor potential mucolipin-2 ion channels in gliomas: role in tumor growth and progression.

The Transient Receptor Potential (TRP) superfamily consists of cation-selective and non-selective ion channels playing an important role both in sensory physiology and in physiopathology in several complex diseases including cancers. Among TRP family, the mucolipin (TRPML1, −2, and −3) channels represent a distinct subfamily of endosome/lysosome Ca2+ channel proteins. Loss-of-function mutations in human TRPML-1 gene cause a neurodegenerative disease, Mucolipidosis Type IV, whereas at present no pathology has been associated to human TRPML-2 channels.Herein we found that human TRPML-2 is expressed both in normal astrocytes and neural stem/progenitor cells. By quantitative RT-PCR, western blot, cytofluorimetric and immunohistochemistry analysis we also demonstrated that TRPML-2 mRNA and protein are expressed at different levels in glioma tissues and high-grade glioma cell lines of astrocytic origin. TRPML-2 mRNA and protein levels increased with the pathological grade, starting from pylocitic astrocytoma (grade I) to glioblastoma (grade IV). Moreover, by RNA interference, we demonstrated a role played by TRPML-2 in survival and proliferation of glioma cell lines. In fact, knock-down of TRPML-2 inhibited the viability, altered the cell cycle, reduced the proliferation and induced apoptotic cell death in glioma cell lines. The DNA damage and apoptosis induced by TRPML-2 loss increased Ser139 H2AX phosphorylation and induced caspase-3 activation; furthermore, knock-down of TRPML-2 in T98 and U251 glioma cell lines completely abrogated Akt and Erk1/2 phosphorylation, as compared to untreated cells.Overall, the high TRPML-2 expression in glioma cells resulted in increased survival and proliferation signaling, suggesting a pro-tumorigenic role played by TRPML-2 in glioma progression.