Knockdown of NOB1 expression by RNAi inhibits cellular proliferation and migration in human gliomas

NOB1 (NIN1/RPN12 binding protein 1 homolog), a ribosome assembly factor, is thought to be essential for the processing of the 20S pre-rRNA into the mature 18S rRNA. It is also reported to participate in proteasome biogenesis. However, the contribution of NOB1 gene dysfunction to the pathology of human diseases, such as gliomas, has not been addressed. Here, we detected expression levels of NOB1 mRNA in U251, U87, U373, and A172 cells by quantitative real-time PCR. To analyze the expression levels of NOB1 protein in glioma tissues, we performed immunohistochemistry on 56 pathologically confirmed glioma samples (7 Grade I cases, 19 Grade II cases, 16 Grade III cases, and 14 Grade IV cases). A recombinant lentivirus expressing NOB1 short hairpin RNA (shNOB1) was constructed and infected into U251 and U87-MG human glioma cells. We found that NOB1 mRNA was expressed in all four cell lines. The expression level of the NOB1 protein was significantly higher in high-grade gliomas than in low-grade gliomas. Knockdown of the NOB1 gene resulted in suppression of the proliferation and the colony-forming abilities of U251 and U87-MG cells, cell cycle arrest during the G₀/G₁ phase, and a significant enhancement of cell apoptosis. In addition, cell migration was significantly suppressed in U251 and U87-MG cells that were infected with the shNOB1-expressing lentivirus. These results suggest that NOB1 promotes glioma cell growth and migration and could be a candidate for molecular targeting during gene therapy treatments of glioma.     

RLIP76 Depletion Enhances Autophagic Flux in U251 Cells

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.     

Aurantiamide acetate suppresses the growth of malignant gliomas in vitro and in vivo by inhibiting autophagic flux

We aim to investigate the effect of aurantiamide acetate isolated from the aerial parts of Clematis terniflora DC against gliomas. Human malignant glioma U87 and U251 cells were incubated with different concentrations (0–100 μM) of aurantiamide acetate. Aurantiamide acetate greatly decreased the cell viability in a dose‐ and time‐dependent manner. It induced moderate mitochondrial fragmentation and the loss of mitochondrial membrane potential. No significant difference was found in the alternation of other intracellular organelles, although F‐actin structure was slightly disturbed. Apparent ultrastructure alternation with increased autophagosome and autolysosome accumulation was observed in aurantiamide acetate‐treated cells. The expression of LC3‐II was greatly up‐regulated in cells exposed to aurantiamide acetate (P < 0.05 compared with control). The cytoplasmic accumulation of autophagosomes and autolysosomes induced by aurantiamide acetate treatment was confirmed by fluorescent reporter protein labelling. Administration of chloroquine (CQ), which inhibits the fusion step of autophagosomes, further increased the accumulation of autophagosomes in the cytoplasm of U87 cells. Autophagy inhibition by 3‐methyladenine, Bafilomycin A1 or CQ had no influence on aurantiamide acetate‐induced cytotoxicity, whereas autophagy stimulator rapamycin significantly suppressed aurantiamide acetate‐induced cell death. The anti‐tumour effects of aurantiamide acetate were further evaluated in tumour‐bearing nude mice. Intratumoural injection of aurantiamide acetate obviously suppressed tumour growth, and increased number of autophagic vacuoles was observed in tumour tissues of animals receiving aurantiamide acetate. Our findings suggest that aurantiamide acetate may suppress the growth of malignant gliomas by blocking autophagic flux.     

HCMV感染对胶质瘤细胞U87自噬的影响

研究人巨细胞病毒(HCMV)感染对神经胶质瘤U87细胞自噬的影响。通过观察微管相关蛋白1轻链3(LC3)、自噬相关基因Beclin1及其蛋白表达的变化,从而探讨HCMV与神经胶质瘤发生、发展的关系及意义。用HCMV AD169(MOI=5)感染神经胶质瘤U87细胞,同时将未感染HCMV的U87细胞作为对照组。分别在6、12、24、48 h用RT-PCR检测Beclin1的表达,Western-blot和免疫荧光检测Beclin1和LC3编码蛋白的表达,最后用CCK-8检测细胞的增殖活性。结果显示,HCMV感染的U87细胞LC3-II蛋白表达水平逐渐下降(P<0.05);同时,HCMV感染的U87细胞Beclin1基因及蛋白的表达水平也逐渐下降(P<0.01),且HCMV感染U87细胞增殖显著(P<0.01)。以上结果表明,HCMV感染抑制胶质瘤U87细胞自噬,并会引起Beclin1表达水平下调,进而导致胶质瘤细胞增殖。     

Chemoresistance to temozolomide in human glioma cell line U251 is associated with increased activity of O6-methylguanine-DNA methyltransferase and can be overcome by metronomic temozolomide regimen

Temozolomide (TMZ) is a novel cytotoxic alkylating agent for chemotherapy of malignant gliomas. However, intrinsic or acquired resistance to TMZ often defines poor efficacy of chemotherapy in malignant gliomas. A growing number of studies indicate that expression of O ⁶-methylguanine-DNA methyltransferase (MGMT) is one of the principal mechanisms responsible for this chemoresistance. In the present study, we evaluated the relationship between expression of MGMT and resistance to TMZ. We generated a TMZ-resistant cell line, U251/TR, by stepwise (8 months) exposure of parental U251 cells to TMZ. The resistance to TMZ was quantified using SRB assay. MGMT expression was evaluated at mRNA (RT-PCR) and protein (Western blot) levels. U251/TR cells showed increased (~ sevenfold) resistance to TMZ. The MGMT expression (both mRNA and protein) was significantly (P < 0.01) increased in U251/TR cells compared with parental U251 cells. Further, MGMT expression fluctuated during exposure of U251/TR cells to TMZ. The resistance of U251/TR cells to TMZ could be overcome by application of elevated doses of TMZ when MGMT expression was at the lowest level. In conclusion, our results demonstrate that the primary mechanism responsible for resistance of U251/TR cells to TMZ is associated with increased expression of MGMT. Resistance of malignant gliomas to TMZ can be overcome by synchronizing metronomic TMZ regimen with MGMT expression.     

Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma

Background

While neurosphere- as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified.

Methodology/Principal Findings

Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFRα, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRα, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype.

Conclusions/Significance

This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas.     

P2X7 receptor antagonism inhibits tumour growth in human high-grade gliomas

Gliomas, the most common primary brain cancer, are highly infiltrative and extremely difficult to treat. Despite advancements, current treatment is limited, with patients surviving for a median of 14–15 months post-diagnosis. Previous research has demonstrated the upregulation of a purinergic receptor, P2X7R, in human gliomas. P2X7R is expressed on both glioma cells and microglia within the glioma microenvironment. It is hypothesized that P2X7R contributes to tumour growth and proliferation via immune-mediated mechanisms involving tumour cells and surrounding microglia. We sought to elucidate the role of P2X7R in a human glioblastoma cell line (U251) and on surgically resected human glioma samples. We treated U251 and human glioma cultures for 72 h with P2X7R antagonists, Brilliant Blue G (BBG), oxidized ATP (oATP) and AZ10606120. Cell counting via fluorescence confocal microscopy was conducted to assess tumour proliferation. We observed no significant reductions in tumour cell numbers following P2X7R antagonism with BBG (20 μM) and oATP (250 μM) in both U251 cells and human glioma samples. Interestingly, there was a significant reduction in tumour cell number in both U251 cells (p =?0.0156) and human glioma samples (p =?0.0476) treated with varying concentrations of AZ10606120. When compared with the conventional chemotherapeutic agent, temozolomide, AZ10606120 was also found to more effectively inhibit tumour proliferation in U251 cells (p <?0.0001). Our pilot results demonstrate a potential trophic role of P2X7R where its inhibition by AZ10606120, a potent antagonist, hinders glioma growth directly or through the inactivation of microglia. This sheds new light on P2X7R as a therapeutic target for human gliomas.

     

Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.     

TRPM2-AS inhibits the growth,migration, and invasion of gliomas through JNK,c-Jun,and RGS4

Gliomas are a group of brain cancers with high mortality and morbidity. Understanding the molecular mechanisms is important for the prevention or treatment of gliomas. The present study was to investigate the effects and mechanisms of long noncoding RNA TRPM2-AS in gliomas proliferation, migration, and invasion. We first compared the levels of TRPM2-AS in 111 patients with glioma to that of the normal control group by a quantitative polymerase chain reaction. The results indicated a significant increase of TRPM2-AS in patients with glioma (2.43 folds of control, p = .0135). MTT methods, wound healing assays, transwell analysis, and clone formation analysis indicated the overexpression of TRPM2-AS promoted the proliferation, migration, and invasion of U251 and U87 cells, while downregulation of TRPM2-AS inhibited the cell proliferation, migration, and invasion significantly (p < .05). To further uncover the mechanisms, bioinformatics analysis was conducted on the expression profiles, GSE40687 and GSE4290, from the Gene Expression Omnibus database. One hundred fifty-six genes were differentially expressed in both datasets (FC > 2.0; p = .05). Among these differentially expressed genes, the level of RGS4 messenger RNA was drastically regulated by TRPM2-AS. Further western-blot analysis indicated the increase of RGS4 protein expression and decrease of p-JNK/JNK and p-c-Jun/c-Jun ratio after TRPM2-AS overexpression. On the other hand, inhibition of TRPM2-AS by small interfering RNA suppressed the expression of RGS4 and promoted the ratios of p-JNK/JNK and p-c-Jun/c-Jun. The present work indicated the mechanisms of the participation of TRPM2-AS in the progression of gliomas might, at least partly, be related to JNK, c-Jun, and RGS4. Our work provided new insights into the underlying mechanisms of glioma cellular functions.     

EphA2对神经胶质瘤细胞系U251的凋亡﹑增殖﹑迁移和侵袭的研究

为了研究EphA2对神经胶质瘤细胞系U251在增殖、凋亡、迁移和侵袭方面所起的作用,用RT-PCR方法检测正常脑组织标本与两种恶性胶质瘤细胞系中EphA2 mRNA表达水平,然后用化学合成的针对EphA2基因的小干扰RNA(siRNA)下调该基因的表达,以检测其在U251中的生物学功能．证实了EphA2基因在正常脑组织标本中的表达水平远低于两种恶性胶质瘤细胞系．把体外化学合成针对EphA2基因的小干扰RNA(siRNA- EphA2)转染入U251细胞后,Western blot, 实时定量 RT-PCR检测到U251细胞中EphA2蛋白及mRNA表达水平都明显降低,并且细胞增殖受到显著抑制,同时出现了明显的细胞凋亡．伤口愈合实验(检测细胞迁移能力),Transwell小室实验(检测细胞侵袭能力)均表明,下调EphA2的表达后,细胞的迁移和侵袭能力较阴性对照组显著减弱．上述结果表明,在神经胶质瘤U251细胞中,EphA2与其恶性增殖及高度侵染性相关,可作为分子治疗的有效靶点．     

Glioma: Application of Whole-Tumor Texture Analysis of Diffusion-Weighted Imaging for the Evaluation of Tumor Heterogeneity

Background and Purpose

To apply a texture analysis of apparent diffusion coefficient (ADC) maps to evaluate glioma heterogeneity, which was correlated with tumor grade.

Materials and Methods

Forty patients with glioma (WHO grade II (n = 8), grade III (n = 10) and grade IV (n = 22)) underwent diffusion-weighted imaging (DWI), and the corresponding ADC maps were obtained. Regions of interest containing the lesions were drawn on every section of the ADC map containing the tumor, and volume-based data of the entire tumor were constructed. Texture and first order features including entropy, skewness and kurtosis were derived from the ADC map using in-house software. A histogram analysis of the ADC map was also performed. The texture and histogram parameters were compared between low-grade and high-grade gliomas using an unpaired student’s t-test. Additionally, a one-way analysis of variance analysis with a post-hoc test was performed to compare the parameters of each grade.

Results

Entropy was observed to be significantly higher in high-grade gliomas than low-grade tumors (6.861±0.539 vs. 6.261±0.412, P  = 0.006). The fifth percentiles of the ADC cumulative histogram also showed a significant difference between high and low grade gliomas (836±235 vs. 1030±185, P = 0.037). Only entropy proved to be significantly different between grades III and IV (6.295±0.4963 vs. 7.119±0.3165, P<0.001). The diagnostic accuracy of ADC entropy was significantly higher than that of the fifth percentile of the ADC histogram (P = 0.0034) in distinguishing high- from low-grade glioma.

Conclusion

A texture analysis of the ADC map based on the entire tumor volume can be useful for evaluating glioma grade, which provides tumor heterogeneity.     

Effect of Bone Morphogenetic Protein 4 in the Human Brain Glioma Cell Line U251

The role of bone morphogenetic protein 4 (BMP4) in gliomas is not clear. We hypothesized that BMP4 inhibits proliferation in the brain glioma cell line U251 through a signaling pathway involving BMP4 and the mothers against decapentaplegic homolog 4 (SMAD4) protein. We exposed U251 cells to Adriamycin (1 g) for 48 h; cell proliferation (MTT assay), expression of BMP4 and SMAD4 (mRNA: qPCR; protein: Western blot) were studied. We further altered expression of BMP4 by overexpression or siRNA silencing, and documented cell responses to Adriamycin. Proliferation of U251 cells was significantly inhibited upon exposure to Adriamycin. This inhibition was associated with increased expression of BMP4. Further, proliferation of U251 cells was inhibited when BMP4 was overexpressed. BMP4 expression negatively correlated with expression of SMAD4, such that elevated levels of BMP4 were associated with decreased expression of SMAD4 and vice versa. The Adriamycin-induced inhibition of proliferation of U251 cells was attenuated when BMP4 was knocked down by siRNA. To conclude, BMP4 is associated with inhibition of proliferation of U251 cells; the effects of BMP4 involve the BMP4-Smad signaling pathway. BMP4 has a potential as a target for glioma therapy.     

WITHDRAWN: Endosomal trafficking of EGFR regulated by hVps18 via interaction of MVB sorting machinery

Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas.     

Nuclear factor-κB signaling pathway is involved in phospholipase Cε-regulated proliferation in human renal cell carcinoma cells

Glioblastoma is the most aggressive cerebral gliomas. Despite advances in therapies, the prognosis is still very poor. Therefore, novel therapeutic strategies are required. As a proteasome inhibitor, bortezomib has shown its efficacy as an active antitumor agent against a variety of tumors. However, inhibition of proteasome activity leads to cell death and also induces cell autophagy, and due to the dual roles of autophagy in the survival and death of tumor cells, the effect of inhibition of autophagy on glioblastoma cells remains to be explored. We therefore assessed whether bortezomib is capable of inducing autophagy, and investigated the antitumor effect of bortezomib combined with autophagy inhibitors on human glioblastoma U251 and U87 cells. Cell viability was measured by MTT assay. The expressions of autophagy and apoptosis-related proteins were determined by Western blot analysis. U251 and U87 cells proliferation was inhibited in a dose-dependent manner. Both apoptosis and autophagy induced by bortezomib were observed in human glioblastoma U87 and U251 cells. However, when U251 and U87 cells were co-treated with bortezomib and autophagy inhibitors 3-MA or Atg7 siRNA, the autophagy inhibitors blocked the autophagy in the cells and resulted in a further inhibition of cell proliferation and a further increase in cell apoptosis as compared with that treated with bortezomib alone. These findings indicated that combination of bortezomib and autophagy inhibitors may shed new light on glioblastoma treatment.     

Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas

Background

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.

Methodology/Principal Findings

In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.

Conclusions/Significance

In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.