RNA interference targeting survivin exerts antitumoral effects in vitro and in established glioma xenografts in vivo

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes-cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI(nu/nu) mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.     

和厚朴酚对组织因子途径抑制物 2诱导神经胶质瘤细胞凋亡的实验研究#br#

目的探讨和厚朴酚对组织因子途径抑制物 2（TFPI 2）表达的影响,以及和厚朴酚和TFPI 2过表达对人胶质瘤U251细胞凋亡的影响。 方法体外培养胶质瘤U87、U251、LN18、LN229、A172细胞系,荧光定量PCR（QPCR）检测不同细胞系中TFPI 2的表达。不同浓度（0、10、20、30、40和50 μmol／L）和厚朴酚干预U251细胞,QPCR和Western blotting检测TFPI 2 mRNA和蛋白的表达。采用腺病毒载体pGSadeno TFPI 2过表达U251细胞内TFPI 2的表达,流式细胞术及caspase 3试剂盒检测细胞凋亡率和caspase 3活性。 结果在选取的5种不同胶质瘤细胞株中,U251和A172细胞内TFPI 2 mRNA明显下降（P＜005）。和厚朴酚呈浓度依赖性增加U251细胞内TFPI 2 mRNA水平;其中50 μmol／L和厚朴酚干预24 h后, TFPI 2 mRNA表达水平升高最明显（P＜005）。腺病毒感染U251细胞后TFPI 2表达水平明显增加（P＜005）。过表达TFPI 2和50 μmol／L和厚朴酚干预均可增加U251细胞凋亡水平和caspase 3的活性（P＜005）。 结论和厚朴酚处理可增加U251细胞内TFPI 2的表达;和厚朴酚联合TFPI 2过表达显著诱导胶质瘤细胞凋亡。     

和厚朴酚对组织因子途径抑制物 2诱导神经胶质瘤细胞凋亡的实验研究#br#

目的探讨和厚朴酚对组织因子途径抑制物 2（TFPI 2）表达的影响,以及和厚朴酚和TFPI 2过表达对人胶质瘤U251细胞凋亡的影响。 方法体外培养胶质瘤U87、U251、LN18、LN229、A172细胞系,荧光定量PCR（QPCR）检测不同细胞系中TFPI 2的表达。不同浓度（0、10、20、30、40和50 μmol／L）和厚朴酚干预U251细胞,QPCR和Western blotting检测TFPI 2 mRNA和蛋白的表达。采用腺病毒载体pGSadeno TFPI 2过表达U251细胞内TFPI 2的表达,流式细胞术及caspase 3试剂盒检测细胞凋亡率和caspase 3活性。 结果在选取的5种不同胶质瘤细胞株中,U251和A172细胞内TFPI 2 mRNA明显下降（P＜005）。和厚朴酚呈浓度依赖性增加U251细胞内TFPI 2 mRNA水平;其中50 μmol／L和厚朴酚干预24 h后, TFPI 2 mRNA表达水平升高最明显（P＜005）。腺病毒感染U251细胞后TFPI 2表达水平明显增加（P＜005）。过表达TFPI 2和50 μmol／L和厚朴酚干预均可增加U251细胞凋亡水平和caspase 3的活性（P＜005）。 结论和厚朴酚处理可增加U251细胞内TFPI 2的表达;和厚朴酚联合TFPI 2过表达显著诱导胶质瘤细胞凋亡。     

PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation

Glioblastoma is a severe type of primary brain tumor and its invasion is strongly correlated with the secretion of matrix metalloproteinases (MMPs). To investigate a role of PTEN, a tumor suppressor gene, in the regulation of hyaluronic acid (HA)-induced invasion of glioma cells, we examined the secretion of MMP-9 in various glioma cells with or without a functional PTEN gene. The secretion of MMP-9 in glioma cells lacking functional PTEN (U87MG, U251MG, and U373MG) was induced by HA, although not in wildtype (wt)-PTEN-harboring cells (LN229, LN18, and LN428). In addition, stable expression of wt-PTEN into U87MG cells significantly decreased the secretion of HA-induced MMP-9 and basal levels of MMP-2, inhibiting the activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2, whereas the secretion levels of the tissue inhibitor of metalloproteinase-1 and -2 were increased, finally resulting in the inhibition of invasion by HA in vitro. Ectopic expressions of adenoviral (Ad)-wt-PTEN and -lipid phosphatase-deficient (G129E)-PTEN, but not both protein and -lipid phosphatase-deficient (C124S)-PTEN, reduced MMP-9 secretion and invasion by HA. These results were also confirmed by expressions of Ad-wt-PTEN and Ad-G129E-PTEN in other glioblastoma cells lacking functional PTEN, U251MG, and U373MG. These findings strongly suggest the possibility that PTEN may block HA-induced MMP-9 secretion and invasion through its protein phosphatase activity.     

Mild Heat Shock Induces Autophagic Growth Arrest,But Not Apoptosis in U251-MG and U87-MG Human Malignant Glioma Cells

Although hyperthermia has been used as a treatment of malignant brain tumors, it is not yet clear what is the mechanism of the cell growth inhibition by heat shock, especially by the temperature which has clinically been applied to tumor-brain border-zone, 42-43 degrees C. Therefore, we evaluated the change of U251-MG and U87-MG human malignant glioma cells after 43 degrees C-heat shock comparing with that of 45 degrees C. First, we observed that cell growth was transiently inhibited after 43 degrees C-heat shock for 3 or 5 days, in U251-MG or U87-MG cells, respectively, which was followed by regrowth. During the period of transient growth inhibition, mild G2/M arrest was observed. However, apoptosis was observed in only 2.7% or 1.5%, of 43 degrees C-heated cells, in U251-MG or U87-MG cells, respectively. Instead, transmission electron micrography showed the formation of vacuoles, degeneration of mitochondria, and autophagosomes. Moreover, in the both cell lines, flow-cytometric analysis with acridine orange revealed the induction of acidic vesicle organelles, which was blocked by 3-methyladenine (3-MA), suggesting the involvement of autophagy. Furthermore, while 3-MA did not increase the anti-tumor effect of 43 degrees C-heat shock, bafilomycin A1, another autophagy inhibitor, did significantly enhance the effect in U251-MG cells. Taken together, mild heat shock (43 degrees C for 2 h) causes autophagy and mild G2/M arrest, but does not induce apparent apoptosis in U251-MG and U87-MG glioma cells. Inhibition of autophagy with bafilomycin A1 may increase the anti-tumor efficacy of mild heat shock against some malignant glioma cells.     

DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响

目的:探讨miR-133a在胶质瘤细胞中的表达及DNA甲基化对其的调控机制并初步探究miR-133a对胶质瘤细胞增殖及凋亡的影响。方法:通过RT-PCR检测胶质瘤组织与正常对照脑组织中miR-133a的表达差异;MSP实验检测胶质瘤组织与正常对照脑组织中miR-133a基因甲基化程度;BSP实验检测正常胶质细胞株HEB与胶质瘤细胞株A172、U87、U251中miR-133a基因的甲基化水平;去甲基化试剂AZA与胶质瘤细胞株A172、U87、U251共培养72 h后,RT-PCR检测上述3株胶质瘤细胞中的miR-133a表达变化;MTT及Annexin V-FITC凋亡实验分别检测AZA处理后的3株胶质瘤细胞的增殖与凋亡能力变化。结果:RT-PCR实验表明与正常对照脑组织相比,胶质瘤组织中miR-133a表达显著下降;MSP实验结果显示,与正常对照脑组织相比,胶质瘤组织中miR-133a基因的甲基化水平明显增高;BSP实验结果显示与正常对照胶质细胞株HEB相比,胶质瘤细胞A172、U87、U251中miR-133a基因的甲基化水平显著增加;RT-PCR实验显示与去甲基化试剂AZA共培养72 h后,胶质瘤细胞株A172、U87、U251细胞中miR-133a的表达显著增加。MTT与Annexin V-FITC凋亡实验结果表明,去甲基化试剂AZA处理后,胶质瘤细胞A172、U87、U251的增殖能力明显下降,细胞的凋亡发生率显著增加。结论:胶质瘤细胞中DNA甲基化通过调控miR-133a的表达而沉默后者发挥抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的功能。     

MK886-induced apoptosis depends on the 5-LO expression level in human malignant glioma cells

Mounting evidence suggests that lipoxygenase (LO)-catalyzed products may play a key role in the development and progression of human cancers. In this study, we analyzed the effects of a 5-LO inhibitor, which inhibits the conversion of arachidonic acid to leukotrienes, on cell proliferation and apoptosis in human malignant glioma cells, including 5-LO-expressing cells U-87MG, A172 and 5-LO non-expressing cell U373. Growth of U-87MG and A172 cells, but not that of U373 cells, was inhibited in a dose-dependent manner by treatment with MK886. Similarly, specific 5-LO silencing by small interfering RNA reduced the growth of U-87MG and A172 cells. MK886 treatment reduced 5-LO activity independently of 5-LO-activating protein (FLAP) in human malignant glioma cells. MK886 treatment also induced cell apoptosis, measured by DNA fragmentation and nuclear condensation, in U-87MG and A172 cells but there were no signs in U373 cells. Moreover, this treatment reduced ERKs phosphorylation and anti-apoptotic molecule Bcl-2 expression, and increased Bax expression in U-87MG and A172 cells. In summary, our results show there is a link between the 5-LO expression status and the extent of MK886-inhibited cell proliferation and apoptosis. Taken together, this study suggest that 5-LO is a possible target for treating patients with gliomas, and 5-LO inhibition might be potent therapy for patients with 5-LO-expressing malignant gliomas.     

外泌体来源的miR-181a促进胶质瘤的血管生成

目的:探讨外泌体来源的miR-181a对胶质瘤血管生成和肿瘤进展的影响.方法:选用2017年8月至2019年12月海南医学院第二附属医院手术切除的83例胶质瘤和13例瘤旁组织标本,以及胶质瘤细胞U87、A172、U251、LN229、U373和小神经胶质细胞HM,用qPCR法检测瘤组织和细胞中miR-181a的表达水平...     

Differential response of human glioblastoma cell lines to combined camptothecin and ionizing radiation treatment

In order to enhance the cytotoxicity of radiation, camptothecin (CPT), an inhibitor of DNA topoisomerase I, was added to the cultured glioma cell lines before irradiation (IR). Radiation responses of five glioblastoma cell lines (U87-MG, U373-MG, GHE, GaMG and SNB-19) treated with CPT were analyzed in terms of cell and colony counts, cell cycle progression, expression of histone gamma H2AX, DNA repair protein Rad50, survivin, cleaved caspase 3, p53 and of topoisomerase I. CPT enhanced the radiotoxicity in U87-MG and SNB-19 cell lines if cell and colony counts were used as the end-points. In contrast, pre-treatment with CPT of U373-MG, GHE and GaMG cell lines did not enhance cytotoxicity of IR in terms of cell and colony counts but accelerated DNA damage repair assessed by Rad50 foci. CPT treated glioma cells revealed at least two subpopulations with respect to the expression of histone gamma H2AX, a marker of DNA double-strand breaks. The cell lines tested also differed in the expression of survivin, cleaved caspase 3, p53 and of topoisomerase I. The failure of CPT to enhance the radiotoxicity of glioma U373-MG, GHE and GaMG cell lines in terms of cell and colony counts was found to correlate with accelerated DNA damage repair, and with low expression of topoisomerase I, a target of CPT.     

Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells

Cyclin-dependent kinase inhibitors (CDKIs) are considered as novel anticancer agents because of their ability to induce growth arrest or apoptosis in tumour cells. It has not yet been fully determined, however, which CDKI is the best candidate for the treatment of malignant gliomas and whether normal brain tissues are affected by CDKI expression. Using recombinant adenoviral vectors that express CDKIs (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)), we compared the antitumour effect of CDKIs on malignant glioma cell lines (A172, GB-1, T98G, U87-MG, U251-MG and U373-MG). p27(KIP1) showed higher ability to suppress the growth of all tumour cells tested than other CDKIs. Interestingly, overexpression of p27(KIP1) induced autophagic cell death, but not apoptosis in tumour cells. On the other hand, p27(KIP1) overexpression did not inhibit the viability of cultured astrocytes (RNB) nor induced autophagy. Overall, our findings suggest that gene transfer of p27(KIP1) may be a promising approach for the therapy of malignant gliomas.     

Adenovirus-mediated overexpression of Fas induces apoptosis of gliomas

Gliomas express a higher amount of Fas than normal brain tissue. It is of interest to know whether expression of the Fas receptor is unfavorable to the antiapoptotic pathways in gliomas. In this study, we introduced the Fas gene via an adenovirus vector (Adeno-Fas) into the A-172, U251, and U-373 MG glioma cell lines, each of which expresses Fas on the cell surface. Infection of Adeno-Fas induced apoptosis in each glioma cell line. In U251 cells and A-172 cells that express the same level of Fas as a result of infection with Adeno-Fas, a much higher percentage of U251 cells underwent apoptosis than did A-172 cells. This suggests that each glioma cell line has its own threshold of Fas expression, above which apoptosis is induced, and that the constitutive expression of Fas is below the level of this threshold. It was found that the constitutive expression of the anti-apoptotic gene Bcl-X(L) is higher in A-172 cells than in U251 cells. Adenovirus-mediated transduction of the Bcl-X(L) gene into U251 cells effectively suppressed Adeno-Fas-mediated apoptosis. These data indicate that the Bcl-X(L) gene is one of the important determinants of the threshold for Fas-mediated apoptosis. When U251 and U-373 MG cells were transduced with the Fas gene controlled by the myelin basic protein promoter, which had been shown to be active in gliomas but not in neural tissues, the cells underwent markedly enhanced apoptosis. Taken together, these results indicate that the overexpression of Fas alone induced apoptosis in each glioma cell line. The degree of Fas-mediated apoptosis was attenuated by the expression of an anti-apoptotic gene, Bcl-X(L). The adenovirus-mediated induction of Fas gene controlled by a tissue-specific promoter (e.g., myelin basic protein promoter) would be a promising therapeutic approach for malignant glioma.     

UHRF2 mRNA Expression is Low in Malignant Glioma but Silencing Inhibits the Growth of U251 Glioma Cells in vitro

UHRF2 is a member of the ubiquitin plant homeo domain RING finger family, which has been proven to befrequently up-regulated in colorectal cancer cells and play a role as an oncogene in breast cancer cells. However,the role of UHRF2 in glioma cells remains unclear. In this study, we performed real-time quantitative PCR on32 pathologically confirmed glioma samples (grade I, 4 cases; grade II, 11 cases; grade III, 10 cases; and gradeIV, 7 cases; according to the 2007 WHO classification system) and four glioma cell lines (A172, U251, U373, andU87). The expression of UHRF2 mRNA was significantly lower in the grade III and grade IV groups comparedwith the noncancerous brain tissue group, whereas its expression was high in A172, U251, and U373 glioma celllines. An in vitro assay was performed to investigate the functions of UHRF2. Using a lentivirus-based RNAinterference (RNAi) approach, we down-regulated UHRF2 expression in the U251 glioma cell line. This downregulationled to the inhibition of cell proliferation, an increase in cell apoptosis, and a change of cell cycledistribution, in which S stage cells decreased and G2/M stage cells increased. Our results suggest that UHRF2may be closely related to tumorigenesis and the development of gliomas.     

Curcumin differentially sensitizes malignant glioma cells to TRAIL/Apo2L-mediated apoptosis through activation of procaspases and release of cytochrome c from mitochondria

Malignant glioma cells are generally resistant or only weakly sensitive to tumor necrosis factor family of cell death-inducing ligands, including TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L. The chemopreventive activity of polyphenolic compounds present in plant-derived food products has been well recognized in epidemiological studies; however, the mechanism of chemoprevention by these dietary constituents largely remains unknown. Curcumin, the yellow pigment in the spice turmeric, has profound anti-inflammatory activity and exhibits chemopreventive and tumor growth inhibitory activity. In the present study, we investigated whether curcumin sensitizes malignant glioma cell lines U251MG and U87MG to TRAIL-induced apoptosis. Treatment with low concentrations (5-20 microM) of curcumin alone had no effect on the viability of either cell line. At low concentration (5 ng/ml) TRAIL induced cytotoxicity in U251MG cells but not in U87MG cells. Whereas curcumin at subtoxic concentration sensitized U87MG cells to TRAIL-induced cytotoxicity, it had no effect on TRAIL-mediated cytotoxicity in U251MG cells. The combined curcumin and TRAIL treatment enhanced accumulation of hypo-diploid U87MG cells in sub G1 cell cycle phase and induced the cleavage of procaspases-3, -8, -9 and release of cytochrome c from mitochondria. These data indicate that curcumin differentially sensitizes glioma cells to TRAIL-induced apoptosis through the activation of both extrinsic (receptor-mediated) and intrinsic (chemical-induced) pathways of apoptosis. These results define a potential use of curcumin to sensitize glioma cells for TRAIL-mediated immunotherapy.     

Distinct patterns of hypoxic expression of carbonic anhydrase IX (CA IX) in human malignant glioma cell lines

The hypoxia-inducible enzyme carbonic anhydrase IX (CA IX) has recently been discussed as a surrogate marker of tumor hypoxia, an indicator of prognosis and a potential therapeutic target in malignant glioma. To characterize patterns of expression of CA IX in human malignant glioma cells, we studied CA IX protein, CA9 mRNA and hypoxia-inducible factor-1α (HIF-1α) protein levels in U87-MG, U251, U373 and GaMG cells exposed to in vitro hypoxia (1, 6 or 24 h at 5%, 1% or 0.1% O₂). All cell lines displayed a strong hypoxic induction of CA9 mRNA in response to prolonged severe hypoxia with cell-line specific patterns at moderate to mild hypoxia and shorter treatment times. Only U87-MG exhibited a strong constitutive, normoxic expression of CA IX protein without a detectable change under hypoxia. In U251 and GaMG cell lines, a marked induction of CA IX protein in response to severe hypoxia was seen. CA IX changes under severe hypoxia and the inhibitory effect of the glycolysis inhibitor iodoacetate (IAA, 50 μM) on hypoxic CA IX overexpression were paralleled by the results for HIF-1α protein. Therefore, immunohistochemical CA IX staining in human malignant glioma specimens can result from low oxygen concentrations or constitutive, oncogene-related, overexpression both of which may be prognostically relevant.     

Mild hyperthermia plus adenoviral p53 over-expression additively inhibits the viability of human malignant glioma cells.

Adenoviral replacement of the p53 gene has already been proved effective for the treatment of various tumours, including malignant gliomas. However, it is difficult to treat malignant glioma with p53 gene therapy alone because of problems with resistance or a less-than-satisfactory response to the treatment. This study investigated whether heat shock at 43 degrees C (mild hyperthermia) augments the cytotoxic effect of p53 gene transfer on malignant glioma cells expressing wild-type p53 (D54) or mutant p53 (U373-MG and U251-MG). The combination of mild hyperthermia and adenoviral p53 over-expression had an additive inhibitory effect on cellular proliferation in all three cell lines studied. Further, both cell cycle analysis and a DNA fragmentation assay showed that apoptosis was induced by p53 over-expression alone but not by heat shock at 43 degrees C alone. However, p53 over-expression followed by mild hyperthermia additively increased the proportion of cells in which apoptosis was induced, regardless of the endogenous p53 status of the tumour cells. Interestingly, a caspase-independent mechanism was observed to be involved in the p53-induced apoptosis in U251-MG and D54 cells. Taken together, the findings showed that combining adenoviral p53 transfer with mild hyperthermia inhibits the proliferation of malignant glioma cells in an additive manner, irrespective of their endogenous p53 status, suggesting a novel treatment strategy for this malignancy.     

Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAP

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in many glioma cell lines. However, treatment with TRAIL in combination with subtoxic doses of roscovitine, a specific inhibitor of Cdc2 and Cdk2, induced rapid apoptosis in TRAIL-resistant glioma cells. Roscovitine could sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells, but not human astrocytes, to TRAIL-induced apoptosis, offering an attractive strategy for safely treating resistant gliomas. Treatment with roscovitine significantly inhibited Cdc2 activity, and expression of a dominant-negative Cdc2 mutant sensitized glioma cells to TRAIL-induced apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in U87MG and T98 glioma cells, treatment with roscovitine recovered TRAIL-induced activation of caspases very efficiently in these cells. We found that treatment with roscovitine or expression of a dominant-negative Cdc2 mutant downregulated the protein levels of survivin and XIAP, two major caspase inhibitors. Overexpression of survivin or XIAP attenuated the apoptosis induced by roscovitine and TRAIL. Taken together, these results suggest that downregulation of survivin and XIAP by subtoxic doses of roscovitine contributes to the amplification of caspase cascades, thereby overcoming glioma cell resistance to TRAIL-mediated apoptosis.