U87 Glioma Cells Research Articles

LY6K promotes glioblastoma tumorigenicity via CAV-1-mediated ERK1/2 signaling enhancement.

Lymphocyte antigen 6 complex, locus K (LY6K) is a putative oncogene in various cancers. Elevated expression of LY6K is correlated with poor patient prognosis in glioblastoma (GBM). The aim of this study is to advance our understanding of the mechanism by which LY6K contributes to GBM tumor biology. Bioinformatic data mining was used to investigate LY6K expression in relation to GBM clinical outcome. To understand the role of LY6K in GBM, we utilized patient-derived glioma stemlike cells (GSCs) and U87 cells and employed immunoblotting, immunofluorescent staining, radiation treatment, and orthotopic GBM xenograft models. Our results show that increased expression of LY6K inversely correlates with GBM patient survival. LY6K promotes tumorigenicity in GBM cells both in vitro and in vivo. The mechanism underlying this tumorigenic behavior is enhancement of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Interestingly, we observed that tumor-promoting LY6K-ERK1/2 signaling is mediated by the interaction of LY6K with caveolin-1, rather than through oncogenic receptor tyrosine kinase-mediated signaling. Moreover, association of LY6K with the cell membrane is crucial for its tumorigenic functions. Finally, DNA methylation maintains LY6K silencing, and hypomethylation of the LY6K promoter increases its expression. In GSCs, ionizing radiation leads to demethylation of the LY6K promoter, thereby increasing LY6K expression and GSC resistance to radiation. Our study highlights the importance of the contribution of LY6K to GBM tumor biology and suggests LY6K as a potential membrane target for treating GBM.

Tumor associated macrophages and its nanovesicles: Immunological insights in cerebral glioma

Purpose: The immune system has a key role in glioma progression, especially the tumor associated macrophages (TAMs). In-vivo, we aimed to study the total TAMs and differential M1 and M2 TAM infiltration in low grade (LGG) versus high grade gliomas (HGG). Also, we investigated the implication of total TAMs and differential M1 and M2 TAMs infiltration on glioma progression. In-vitro, we studied the effect of soluble factors present in nanovesicles (NV) released from M1 TAMs on the fate of glioma cells.Methods: In-vivo, we performed immunohistochemistry using iNOS and CD163 (markers for M1 and M2 respectively). In-vitro, we polarized the human monocytes U937 cell line into M1, we isolated the NV from the M1-conditioned medium (CM) by centrifugation and filtration; then, the protein content of the NV was quantified by the protein assay. We added M1-NV on U251 glioma cells and we studied the cellular activation of glioma cells using the MTT assay. To assess the apoptosis of U251, we used the flow-cytometry. Apoptotic cells were identified by annexin V and Propidium Iodide (markers for early and late apoptosis respectively).Results: in-vivo, there is an M1/M2 imbalance in early stages of glioma which is associated with earlier progression to high malignancy. Also, the higher M2 infiltration, the earlier is the progression. In-vitro, M1-NV had a more potent anti-tumor effect compared to its corresponding CM. We assume that our experimental results can be a future treatment for the cerebral glioma.

A role of metallothionein-3 in radiation-induced autophagy in glioma cells

Although metallothionein-3 (MT3), a brain-enriched form of metallothioneins, has been linked to Alzheimer’s disease, little is known regarding the role of MT3 in glioma. As MT3 plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of MT3 with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by MT3 knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, MT3 knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that MT3 and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.

Effect and mechanism of YB-1 knockdown on glioma cell growth, migration, and apoptosis.

Y-box binding protein 1 (YB-1) is manifested as its involvement in cell proliferation and differentiation and malignant cell transformation. Overexpression of YB-1 is associated with glioma progression and patient survival. The aim of this study is to investigate the influence of YB-1 knockdown on glioma cell progression and reveal the mechanisms of YB-1 knockdown on glioma cell growth, migration, and apoptosis. It was found that the knockdown of YB-1 decreased the mRNA and protein levels of YB-1 in U251 glioma cells. The knockdown of YB-1 significantly inhibited cell proliferation, colony formation, and migration in vitro and tumor growth in vivo. Proteome and phosphoproteome data revealed that YB-1 is involved in glioma progression through regulating the expression and phosphorylation of major proteins involved in cell cycle, adhesion, and apoptosis. The main regulated proteins included CCNB1, CCNDBP1, CDK2, CDK3, ADGRG1, CDH-2, MMP14, AIFM1, HO-1, and BAX. Furthermore, it was also found that YB-1 knockdown is associated with the hypo-phosphorylation of ErbB, mTOR, HIF-1, cGMP-PKG, and insulin signaling pathways, and proteoglycans in cancer. Our findings indicated that YB-1 plays a key role in glioma progression in multiple ways, including regulating the expression and phosphorylation of major proteins associated with cell cycle, adhesion, and apoptosis.

MicroRNA-519d-3p inhibits cell proliferation and cell cycle G1/S transition in glioma by targeting CCND1.

Glioma is the most common highly malignant primary brain tumor. MicroRNA-519d-3p exerts important effects in several tumors, but its functional role in glioma remained poorly understood. In this study, we found miR-519d-3p expression was significantly decreased in glioma tissues and cell lines. Moreover, the in vitro experiments showed that overexpression of miR-519d-3p suppressed cell proliferation and induced cell cycle G0/G1 phase arrest using MTT and flow cytometry assays in glioma cell lines, U87 and U251. Mechanistically, Cyclin D1 (CCND1) was predicted and confirmed as the direct target genes of miR-519d-3p using luciferase report assay. In addition, knockdown of CCND1 imitated the suppressive effects of miR-519d-3p on cell proliferation and cell cycle progression. Furthermore, restoration of CCND1 reversed the effects of miR-519d-3p overexpression in glioma cells. Taken together, these data demonstrate that suppression of CCND1 by miR-519d-3p might be a therapeutic target for glioma.Abbreviations miR-519d-3p: microRNA-519d-3p; CCND1: Cyclin D1; ATCC: American Type Culture Collection; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PI: propidium iodide; WT: wild type; MUT: mutant type; SD: standard deviation.

LncRNA CCAT2 Enhanced Resistance of Glioma Cells Against Chemodrugs by Disturbing the Normal Function of miR-424.

BackgroundAggressive metastasis of tumor cells assumed a constructive role in strengthening chemoresistance of tumors, so this investigation was intended to elucidate if lncRNA CCAT2 sponging downstream miR-424 regulated chemotolerance of glioma cells by boosting metastasis of glioma cells.MethodsOne hundred and twenty-eight pairs of glioma tissues and corresponding adjacent tissues were resected from glioma patients during their operation, and we also purchased a series of glioma cell lines, including U251, U87, A172 and SHG44. Furthermore, pcDNA3.1-CCAT2, si-CCAT2, miR-424 mimic and miR-424 inhibitor were transfected into SHG44 and U251 cell lines, so as to evaluate impacts of CCAT2 and miR-424 on chemosensitivity of the glioma cells. Besides, proliferation, invasion and metastasis of the cells were determined through the implementation of colony formation assay, transwell assay and scratch assay.ResultsGlioma tissues and cells were monitored with higher CCAT2 expression and lower miR-424 expression than adjacent normal tissues and NHA cell line (P<0.05). Among the glioma cell lines, the SHG44 cell line showed the strongest resistance against teniposide, temozolomide and cisplatin (P<0.05), whereas the U251 cell line was more sensitive to teniposide, temozolomide, vincristine and cisplatin than any other cell line (P<0.05). Besides, pcDNA3.1-CCAT2 and miR-424 inhibitor could enhance tolerance of glioma cell lines against drugs (P<0.05). Moreover, in-vitro transfection of si-CCAT2 and miR-424 mimic could significantly retard proliferation, invasion and migration of SHG44 and U251 cells (P<0.05), and CCAT2 was found to negatively regulate miR-424 expression by sponging it (P<0.05). In addition, CHK1 was deemed as the molecule targeted by upstream miR-424, and its overexpression can changeover the effects of miR-424 mimic on proliferation and metastasis of SHG44 and U251 cells.ConclusionlncRNA CCAT2/miR-424/Chk1 axis might serve as a promising target for improving chemotherapeutic efficacies in glioma treatment.

Fluorescence-guided stereotactic biopsy: a proof-of-concept study

OBJECTIVEAccurate histopathological diagnoses are often necessary for treating neuro-oncology patients. However, stereotactic biopsy (STB), a common method for obtaining suspicious tissue from deep or eloquent brain regions, fails to yield diagnostic tissue in some cases. Failure to obtain diagnostic tissue can delay initiation of treatment and may result in further invasive procedures for patients. In this study, the authors sought to determine if the coupling of in vivo optical imaging with an STB system is an effective method for identification of diagnostic tissue at the time of biopsy.METHODSA minimally invasive fiber optic imaging system was developed by coupling a 0.65-mm-diameter coherent fiber optic fluorescence microendoscope to an STB system. Human U251 glioma cells were transduced for stable expression of blue fluorescent protein (BFP) to produce U251-BFP cells that were utilized for in vitro and in vivo experiments. In vitro, blue fluorescence was confirmed, and tumor cell delineation by fluorescein sodium (FNa) was quantified with fluorescence microscopy. In vivo, transgenic athymic rats implanted with U251-BFP cells (n = 4) were utilized for experiments. Five weeks postimplantation, the rats received 5–10 mg/kg intravenous FNa and underwent craniotomies overlying the tumor implantation site and contralateral normal brain. A clinical STB needle containing our 0.65-mm imaging fiber was passed through each craniotomy and images were collected. Fluorescence images from regions of interest ipsilateral and contralateral to tumor implantation were obtained and quantified.RESULTSLive-cell fluorescence imaging confirmed blue fluorescence from transduced tumor cells and revealed a strong correlation between tumor cells quantified by blue fluorescence and FNa contrast (R2 = 0.91, p &lt; 0.001). Normalized to background, in vivo FNa-mediated fluorescence intensity was significantly greater from tumor regions, verified by blue fluorescence, compared to contralateral brain in all animals (301.7 ± 34.18 relative fluorescence units, p &lt; 0.001). Fluorescence intensity measured from the tumor margin was not significantly greater than that from normal brain (p = 0.89). Biopsies obtained from regions of strong fluorescein contrast were histologically consistent with tumor.CONCLUSIONSThe authors found that in vivo fluorescence imaging with an STB needle containing a submillimeter-diameter fiber optic fluorescence microendoscope provided direct visualization of neoplastic tissue in an animal brain tumor model prior to biopsy. These results were confirmed in vivo with positive control cells and by post hoc histological assessment. In vivo fluorescence guidance may improve the diagnostic yield of stereotactic biopsies.

Heat Shock Cognate Protein 70 Enhanced Integrin β1 Mediated Invasion in Cancer Cells.

PurposeGlioblastoma is one of the most common malignant cancers worldwide. In our previous work, we have shown that heat shock cognate protein 70 (Hsc70) functions as a positive growth regulator in glioma. We investigated the role of Hsc70 in integrin β1 mediated invasion of glioma cells.MethodsIn order to investigate whether the down-regulation of Hsc70 would affect the expression of integrin β1 subunit, HeLa cells were transiently transfected with Hsc70-AS or pcDNA3.0 vectors and the down-regulation of Hsc70 was confirmed by Western blotting. Human brain glioma U87 cells were stably transfected with Hsc70-AS or pcDNA3.0 vectors to further elucidate the relationship between Hsc70 and integrin β1 in human glioma cells. Cellular localization of integrin β1 was detected using immunofluorescence confocal microscopy analysis.ResultsHere we reported that down-regulation of the expression of Hsc70 in U87 cells by transfection with antisense cDNA specifically increased the expression of cell surface integrin β1 without changing its mRNA. Meanwhile, the integrin β1 125-kD mature form increased while 105-kD precursor form decreased when Hsc70 was down-regulated. Mechanically, the U87 cells transfected with antisense cDNA of Hsc70 decreased the Golgi localization of integrin β1, strengthened its interaction with integrin α5 subunit, and enhanced the adhesion ability to fibronectin (FN) and the phosphorylation level of focal adhesion kinase (FAK).ConclusionOverall, these results suggested that the down-regulation of Hsc70 expression could promote the expression of cell surface integrin β1 and subsequently inhibit glioma invasion phenotype.

MiR-1254 inhibits progression of glioma in vivo and in vitro by targeting CSF-1.

The role of miRNAs (microRNAs) has been implicated in glioma initiation and progression, although the inherent biochemical mechanisms still remain to be unravelled. This study strived to evaluate the association between CSF‐1 and miR‐1254 and their effect on advancement of glioma cells. The levels of miR‐1254 in glioma cells and tissues were determined by real‐time RT‐PCR. Proliferation, apoptosis and cell cycle arrest, invasion and migration, were assessed by CCK‐8 assay, colony formation assay, flow cytometry, transwell assay and wound‐healing assay, respectively. The targeted relationship between miR‐1254 and CSF‐1 was confirmed by dual‐luciferase reporter assay. The effects of CSF‐1 on cellular functions were also assessed. The in vivo effect of miR‐1254 on the formation of a tumour was explored by using the mouse xenograft model. We found in both glioma tissues and glioma cells, the down‐regulated expressions of miR‐1254 while that of CSF‐1 was abnormally higher than normal level. The target relationship between CSF‐1 and miR‐1254 was validated by dual‐luciferase reporter assay. The CSF‐1 down‐regulation or miR‐1254 overexpression impeded the invasion, proliferation and migratory ability of U251 and U87 glioma cells, concurrently occluded the cell cycle and induced cell apoptosis. Moreover, in vivo tumour development was repressed due to miR‐1254 overexpression. Thus, CSF‐1 is targeted directly by miR‐1254, and the miR‐1254/CSF‐1 axis may be a potential diagnostic target for malignant glioma.

Endoplasmic Reticulum Stress Contributes to Indomethacin-Induced Glioma Apoptosis.

The dormancy of cellular apoptotic machinery has been highlighted as a crucial factor in therapeutic resistance, recurrence, and poor prognosis in patients with malignancy, such as malignant glioma. Increasing evidence indicates that nonsteroidal anti-inflammatory drugs (NSAIDs) confer chemopreventive effects, and indomethacin has been shown to have a novel chemotherapeutic application targeting glioma cells. To extend these findings, herein, we studied the underlying mechanisms of apoptosis activation caused by indomethacin in human H4 and U87 glioma cells. We found that the glioma cell-killing effects of indomethacin involved both death receptor- and mitochondria-mediated apoptotic cascades. Indomethacin-induced glioma cell apoptosis was accompanied by a series of biochemical changes, including reactive oxygen species generation, endoplasmic reticulum (ER) stress, apoptosis signal-regulating kinase-1 (Ask1) activation, p38 hyperphosphorylation, protein phosphatase 2A (PP2A) activation, Akt dephosphorylation, Mcl-1 and FLICE-inhibiting protein (FLIP) downregulation, Bax mitochondrial distribution, and caspases 3/caspase 8/caspase 9 activation. Data on pharmacological inhibition related to oxidative stress, ER stress, free Ca2+, and p38 revealed that the axis of oxidative stress/ER stress/Ask1/p38/PP2A/Akt comprised an apoptotic cascade leading to Mcl-1/FLIP downregulation and glioma apoptosis. Since indomethacin is an emerging choice in chemotherapy and its antineoplastic effects have been demonstrated in glioma tumor-bearing models, the findings further strengthen the argument for turning on the aforementioned axis in order to activate the apoptotic machinery of glioma cells.

Silencing lncRNA GIHCG increases radiosensitivity of glioma cells by up-regulating miR-146a-3p

Objective To investigate the effect of lncRNA GIHCG on the radiosensitivity of glioma cells and its mechanism. Methods The expression levels of GIHCG and miR-146a-3p in human brain normal glial cells HEB and glioma cell lines U251, A172, SHG139 and U87 were quantitatively measured by qRT-PCR assay. U251 and SHG139 cells were used for subsequent experiment. After silencing the expression of GIHCG or overexpressing miR-146a-3p in U251 and SHG139 cells, cell proliferation was detected by MTT assay, cell apoptosis was detected by flow cytometry, cell radiosensitivity was detected by colony formation assay and the expression levels of CDK1, CyclinD1, Bcl-2 and Bax proteins were measured by Western blot. The bioinformatics software predicted the presence of a binding site for GIHCG and miR-146a-3p. Dual luciferase reporter gene assay and qRT-PCR assay were adopted to verify the targeting relationship between GIHCG and miR-146a-3p. Results Compared with HEB cells, the expression of GIHCG was significantly up-regulated in glioma U87, U251, A172 and SHG139 cells (all P<0.05), whereas that of miR-146a-3p was remarkably down-regulated (P<0.05). Silencing GIHCG expression or overexpression of miR-146a-3p significantly decreased the U251 and SHG139 cell survival rate, survival fraction and the expression of CDK1, CyclinD1 and Bcl-2 proteins (all P<0.05), whereas considerably increased the apoptotic rate and expression of Bax protein (both P<0.05). GIHCG performed targeted negative regulation of miR-146a-3p expression in U251 and SHG139 cells and inhibition of miR-146a-3p expression reversed the effect of silencing GIHCG on proliferation, apoptosis and radiosensitivity of glioma cells. Conclusion Silencing GIHCG expression up-regulates the expression of miR-146a-3p, thereby enhancing the radiosensitivity of glioma cells. Key words: Glioma; lncRNA GIHCG; miR-146a-3p gene; Radiosensitivity

Inhibition of TGF-β Signaling in Gliomas by the Flavonoid Diosmetin Isolated from Dracocephalum peregrinum L.

Background:Dracocephalum peregrinum L., a traditional Kazakh medicine, has good expectorant, anti-cough, and to some degree, anti-asthmatic effects. Diosmetin (3′,5,7-trihydroxy-4′-methoxyflavone), a natural flavonoid found in traditional Chinese herbs, is the main flavonoid in D. peregrinum L. and has been used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic, and anti-inflammatory effects. The present study aimed to investigate the effects of diosmetin on the proliferation, invasion, and migration of glioma cells, as well as the possible underlying mechanisms. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), scratch wound, and Transwell assays were used to demonstrate the effects of diosmetin in glioma. Protein levels of Bcl-2, Bax, cleaved caspase-3, transforming growth factor-β (TGF-β), E-cadherin, and phosphorylated and unphosphorylated smad2 and smad3 were determined by Western blots. U251 glioma cell development and progression were measured in vivo in a mouse model. Results: Diosmetin inhibited U251 cell proliferation, migration, and invasion in vitro, the TGF-β signaling pathway, and Bcl-2 expression. In contrast, there was a significant increase in E-cadherin, Bax, and cleaved caspase-3 expression. Furthermore, it effectively reduced the tumorigenicity of glioma cells and promoted apoptosis in vivo. Conclusion: The results of this study suggest that diosmetin suppresses the growth of glioma cells in vitro and in vivo, possibly by activating E-cadherin expression and inhibiting the TGF-β signaling pathway.

AnvirzelTMregulates cell death through inhibiting GSK-3 activity in human U87 glioma cells

ABSTRACTObjectives: Cardiac glycosides are used as potential anti-cancer agents due to their effects on the inhibition of proliferation and induction of apoptosis and/or autophagy in cancer cells. Herein, we aimed to study the potential signaling pathways taken role in differential cell-death properties of AnvirzelTM which is consisted of two toxic cardiac glycosides (oleandrin and oleandrigenin), in U87 human glioblastoma cells.Methods: The anti-proliferative and anti-migratory effects of AnvirzelTM were assessed in U87 cells by WST-1 assay and wound healing assay, respectively. After treatment of AnvirzelTMwith doses of 10, 25, 50, 100 and 250 μg/ml, expression levels of proteins related to cell death were investigated by Western blot.Results: Anvirzel™ markedly inhibited the growth of U87 cells in a time- and dose-dependent manner following 24 h and 48 h treatments (p < 0.05). In addition, it was found that Anvirzel™ inhibited GSK-3, NOS and HIF1-α expressions whereas activated ERK in U87 cells compared to vehicle (p < 0.05).Discussion: The results suggested that AnvirzelTM regulated cell death distinctly from apoptosis in human glioblastoma cells. Further studies are required for validation of mechanistic insights about the potential signaling pathways taken role in differential cell death properties of AnvirzelTM.

Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo

Background and Aim: Our previous study demonstrated that SLC22A18 downregulation through promoter methylation and Sequence binding protein 1 (SATB1) upregulation are associated with the development and progression of glioma. This study aimed to examine the effect of combined SLC22A18 and short hairpin RNA (shRNA) targeting SATB1 gene therapy on glioma growth and invasion. Materials and Methods: Here, a combined gene therapy to upregulate SLC22A18 and downregulate SATB1 in malignant glioma was evaluated both in vitro and in vivo. Glioma U251 cells overexpressing SLC22A18 and underexpressing SATB1 were generated to investigate the effects of these changes on cell survival, as measured by the methyl thiazol tetrazolium assay, and on cell invasion, as measured by cell invasion and migration assays. In addition, analysis of the cell cycle was performed using flow cytometry in vitro. The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046). Results: The upregulation of SLC22A18 and downregulation of SATB1 significantly inhibited growth and invasion in vitro and reduced in vivo tumor growth of human glioma U251 cells. Furthermore, we revealed that U251 cells were arrested at the G0/G1 phase. Similar data for apoptotic glioma cell death were obtained in tumor cells from an in vivo glioma xenograft model after transfection. At the same total dose, the therapeutic effect was markedly better in the glioma xenografts transfected with both SLC22A18 gene and SATB1 short hairpin RNA (shRNA) expression vectors compared with tumors transfected with either agent alone. The levels of SLC22A18 and SATB1 protein expression were respectively increased and decreased in the glioma cells.Conclusion: These results demonstrate that combination treatment with SLC22A18 gene and SATB1 shRNA expression vectors effectively inhibits the growth of human malignant glioma cells both in vitro and in glioma xenografts in vivo, suggesting a promising novel strategy for glioma therapy that warrants further study.

Silencing of NAMPT leads to up-regulation of insulin receptor substrate 1 gene expression in U87 glioma cells.

The aim of the present study was to investigate the effect of adipokine NAMPT (nicotinamide phosphoribosyltransferase) silencing on the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other proliferation related proteins in U87 glioma cells for evaluation of the possible significance of this adipokine in intergenic interactions. The silencing of NAMPT mRNA was introduced by NAMPT specific siRNA. The expression level of NAMPT, IGFBP3, IRS1, HK2, PER2, CLU, BNIP3, TPD52, GADD45A, and MKI67 genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Anti-visfatin antibody was used for detection of NAMPT protein by Western-blot analysis. It was shown that the silencing of NAMPT mRNA led to a strong down-regulation of NAMPT protein and significant modification of the expression of IRS1, IGFBP3, CLU, HK2, BNIP3, and MKI67 genes in glioma cells and a strong up-regulation of IGFBP3 and IRS1 and down-regulation of CLU, BNIP3, HK2, and MKI67 gene expressions. At the same time, no significant changes were detected in the expression of GADD45A, PER2, and TPD52 genes in glioma cells treated by siRNA specific to NAMPT. Furthermore, the silencing of NAMPT mRNA suppressed the glioma cell proliferation. Results of this investigation demonstrated that silencing of NAMPT mRNA with corresponding down-regulation of NAMPT protein and suppression of the glioma cell proliferation affected the expression of IRS1 gene as well as many other genes encoding the proliferation related proteins. It is possible that dysregulation of most of the studied genes in glioma cells after silencing of NAMPT is reflected by a complex of intergenic interactions and that NAMPT is an important factor for genome stability and regulatory mechanisms contributing to the control of glioma cell metabolism and proliferation.

Nanocrystallized Oleanolic Acid Better Inhibits Proliferation, Migration and Invasion in Intracranial Glioma via Caspase-3 Pathway.

Glioma associates with high malignancy and poor prognosis for traditional treatment. Oleanolic acid (OA) has been confirmed to have an inhibitory effect on different kinds of tumors, while accompanying with low efficiency because of its large molecular mass and low solubility. Nanoliposome is an appropriate drug delivery system that can compensate for the limitations of traditional insoluble drugs, involving improvement of their solubility, stability and lipophilicity. In the present study, we comprised of OA covered with nanoliposomes, named OAnano, to observe antitumor effects on U87 glioma cells. The results showed that OAnano raised the solubility and oil-water partition coefficient. OAnano suppressed proliferation of U87 glioma cells, and also had an anticancer effect on U87 glioma cells, which was found to be higher than that of OA. Moreover, treatment with OAnano induced apoptosis and degraded migration ability by caspase-3 pathway. In conclusion, our results demonstrated that OA covered with nanoliposomes led to enhanced anticancer effects by suppressing proliferation, migration and invasion abilities. The findings may provide a reliable reference for development of new anti-cancer drugs.

The enhanced treatment efficacy of invasive brain glioma by dual-targeted artemether plus paclitaxel micelles

High grade-gliomas are highly invasive and prone to metastasis, leading to poor survival and prognosis. Currently, we urgently need a new treatment strategy to effectively inhibit glioma. In this study, artemether and paclitaxel were used as two agents for tumour suppression. Two functional materials were synthesised and modified on the surface of the micelle as targeting molecules. The addition of two functional materials confers the ability of the micelles to effectively cross the blood-brain barrier (BBB) and then target the glioma cells. Thus, this dual-targeted delivery system allows the drug to play a better role in inhibiting tumour invasion and vasculogenic mimicry (VM) channels. In this paper, the anticancer effects of dual-targeted artemether plus paclitaxel micelles on glioma U87 cells were studied in three aspects: (I) In vitro and in vivo targeting assessment, including the role of penetrating BBB and targeting glioma; (II) In vitro regulation of invasion-associated proteins; (III) Inhibition of VM channels formation and invasion in vitro; (IV) The study of pharmacodynamics in tumour-bearing mice. These results suggest that dual-targeted artemether plus paclitaxel micelle may provide a new strategy to treat glioma via inhibiting invasive and VM channels.

The lncRNA CASC2/miR-18b/RORA Axis Suppresses Invasion and the Epithelial-Mesenchymal Transition in Multifocal Glioblastomas &lt;i&gt;via&lt;/i&gt; the TGF-β1 /Smad Signaling Pathway

Background: Multifocal glioblastoma is an uncommon and refractory subtype of high grade glioma with poor prognosis. The epithelial-mesenchymal transition (EMT) is an important cause of enhanced glioblastoma invasiveness and tumor recurrence. Our previous study found that retinoic acid receptor-related orphan receptor A (RORA) is a nuclear receptor, and plays an important role in inhibiting proliferation and tumorigenesis of glioma. We further confirmed that RORA was downregulated in multifocal glioblastoma (GBM), therefore, we determined whether RORA was involved in the migration, invasion, and EMT of multifocal GBM. Methods: Human glioma cell lines, U87 and T98G, and patient-derived glioma stem cells (GSCs), GSC2C and GSC4D, were used for in vitro and in vivo experiments. The expressions of RORA, miR-18b, and lncRNA CASC2 in glioma cells were detected by RT-qPCR, western blotting. The biological effects of RORA, miR-18b, and CASC2 on glioma migration, invasion, and EMT were evaluated using the migration assay, Transwell, immunofluorescence staining, and xenograft experiments. Results: RORA was expressed at lower levels in multifocal glioblastoma and negatively regulated the migration, invasion, and EMT of multifocal GBM via the TGF-β1/Smad signaling pathway. MiR-18b functioned as a promotor in multifocal glioblastoma, while CASC2 played the role of a tumor suppressor. MiR-18b inhibited RORA expression via binding to its mRNA 3'-UTR in multifocal glioblastoma, while CASC2 upregulated RORA expression via miR-18b-mediated competitive endogenous RNA mechanism. Conclusions: The lncRNA CASC2/miR-18b/RORA/TGF- β1/Smad signaling pathway axis, which lead to the EMT and enhanced invasiveness of multifocal GBM, may be a promising therapeutic strategy for multifocal glioblastoma. Funding Statement: This work was supported by the Major Disease Prevention and Control Technology Action Plan of China (2018ZX-07S-006), the Liaoning BaiQianWan Talents Program (No. 2019-B45), the Social Development Program from Shenyang Science and Technology Bureau, China (20-205-4-075), the Project of JiangSu XianSheng Pharmaceutical Co. (JSXSZD-SA2020-06-004), and the Shanghai Sailing Program (No. 19YF1439000). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Patients and controls were acquired with informed consent, under the protocol approved by the First Hospital of China Medical University research ethics committee.

Valproic Acid Enhanced Apoptosis by Promoting Autophagy Via Akt/mTOR Signaling in Glioma

Glioma is the most common malignant tumor in the central nervous system with a poor median survival. Valproic acid (VPA), a widely used antiepileptic drug, has been found to have antitumor effects on gliomas, but its role still has not been determined. In this study, we investigated VPA-induced apoptotic and autophagic effects on human U251 and SNB19 cells by cell counting kit-8 assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated nick end labeling staining, western blots, and immunofluorescence assay in vitro, and then we further explored the role of autophagy in apoptosis by using the selective antagonist MHY1485. The data showed that VPA inhibited U251 and SNB19 glioma cells viability in a dose-dependent and time-dependent manner and induced apoptosis through the mitochondria-dependent pathway in vitro. In addition, VPA activated the Akt/mTOR pathway by decreasing their protein phosphorylation to promote cellular apoptosis. Surprisingly, the mTOR agonist MHY1485, causing a strong elevation of mTOR activity, partially reduced apoptosis ratio, which supposing that the autophagy of VPA is involved in the regulation of apoptosis. These findings suggest that VPA enhanced apoptosis by promoting autophagy via Akt/mTOR signaling in glioma, which could be further evaluated as a reliable therapy for glioma.

Moxidectin induces Cytostatic Autophagic Cell Death of Glioma Cells through inhibiting the AKT/mTOR Signalling Pathway.

Moxidectin (MOX), a broad-spectrum antiparasitic drug, has been characterized as a potential anti-glioma agent. The main objective of this study was to explore autophagy induced by MOX in glioma U251 and C6 cells, and the deep underlying molecular mechanisms. In addition, the effects of autophagy on apoptosis in glioma cells were tested. Autophagy was measured by transmission electron microscopy (TEM), immunofluorescence, western blot and immunohistochemistry. Cell viability was detected with MTT and colony formation assay. The apoptosis rate was measured by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Additonally, autophagy inhibition was achieved by using 3-Methyladenine (3-MA) and chloroquine (CQ). U251-derived xenografts were established for examination of MOX-induced autophagy on glioma in vivo. Firstly, our research found that MOX stimulated autophagy of glioma cells in a dose-dependent manner. Secondly, we found that MOX induced autophagy by inhibiting the AKT/mTOR signalling pathway. Thirdly, inhibition of autophagy could reduce apoptosis in MOX-treated glioma cells. Finally, MOX induced autophagy, and autophagy increased the apoptosis effect of MOX on U251 in vivo. In conclusion, our data provide evidence that MOX can induce autophagy in glioma cells, and autophagy could increase MOX-induced apoptosis through inhibiting the AKT/mTOR signalling pathway. These findings provided a new prospect for the application of MOX and a novel targeted therapy for the treatment of gliomas.