Arachidonylethanolamide induces apoptosis of human glioma cells through vanilloid receptor-1

The anti-tumor properties of cannabinoids have recently been evidenced, mainly with delta9-tetrahydrocannabinol (THC). However, the clinical application of this drug is limited by possible undesirable side effects due to a broad expression of cannabinoid receptors (CB1 and CB2). An attractive field of research therefore is to identify molecules with more selective tumor targeting. This is particularly important for malignant gliomas, considering their poor prognosis and their location in the brain. Here we investigated whether the most potent endogenous cannabinoid, arachidonylethanolamide (AEA), could be a candidate. We observed that AEA induced apoptosis in long-term and recently established glioma cell lines via aberrantly expressed vanilloid receptor-1 (VR1). In contrast with their role in THC-mediated death, both CB1 and CB2 partially protected glioma against AEA-induced apoptosis. These data show that the selective targeting of VR1 by AEA or more stable analogues is an attractive research area for the treatment of glioma.     

Neurostatin blocks glioma cell cycle progression by inhibiting EGFR activation

The high frequency and malignancy of human glioblastomas has stimulated the search for potential therapeutic approaches. The control of the glioma cell proliferation in response to mitogenic signals is one of the most promising antitumoral strategies, and the main target of several therapies.Neurostatin, an O-acetylated derivative of the ganglioside GD1b, has potent antiproliferative activity over the in vitro and in vivo growth of glioma cells. The mechanism of its antitumoral action is the focus of the present study. Using a combined in vitro–in vivo approach, we observed that neurostatin arrested glioma proliferation by inhibiting the expression of cell cycle promoters (i.e. cyclins and CDKs) and promoting the expression of cell cycle inhibitors (i.e. p21 and p27). Neurostatin inhibits epidermal growth factor receptor (EGFR) signaling pathways, blocking the activation of the main promitogenic MAPKs and PI3K pathways. Neurostatin action not only interferes in the cell cycle progression, but also in the protection from apoptosis, and the generation of angiogenic and invasive responses.The antitumoral actions described here point to neurostatin as a novel and promising chemotherapeutic agent for glioma treatment.     

Single doublecortin gene therapy significantly reduces glioma tumor volume

We employed lentivirus‐based doublecortin (DCX), as a glioma suppressor gene therapy in an intracranial glioma tumor xenograft model in nude rats. Single DCX‐expressing lentivirus was directly administered into the tumor on day 8 after U87 tumor cell implantation. DCX treatment significantly reduced U87 glioma tumor volume (～60%) on day 14 after DCX lentivirus injection and significantly improved median survival of tumor‐bearing nude rats. DCX synthesis induced neuronal markers MAP2, TUJ1, and PSA‐NCAM and the glial marker glial fibrillary acidic protein (GFAP) in the implanted U87 glioma tumors. DCX synthesis induced GFAP that colocalized with tubulin in the mitotic stage, inhibited cleavage furrow during cytokinesis, and blocked mitosis in glioma cells. DCX lentivirus infection did not induce apoptosis but significantly inhibited expression of the proliferation marker Ki‐67 and the blood vessel marker von‐Willebrand factor (vWF). U87 and other glioma cells except for brain tumor stem cells (BTSCs) do not express neuronal markers or both neuronal and glial markers. DCX‐synthesizing glioma cells express a phenotype of antiangiogenic BTSC‐like cells with terminal differentiation that causes remission of glioma cells by blocking mitosis via a novel DCX/GFAP pathway. Direct local delivery of lentivirus‐based DCX gene therapy is a potential differentiation‐based therapeutic approach for the treatment of glioma. © 2009 Wiley‐Liss, Inc.     

4E‐BP1 regulates the sensitivity of human glioma cells to chemotherapy through PI3K/Akt/mTOR‐independent pathway

Drug resistance is one of the most formidable obstacles for treatment of glioma. Eukaryotic initiation factor 4E‐binding protein (4E‐BP1), a key component in the rate‐limiting step of protein translation initiation, is closely associated with poor prognosis in multiple tumor types. However, it is unclear whether 4E‐BP1 is involved in the drug resistance of human glioma. Herein we show that the expression of 4E‐BP1 in human SWOZ2‐BCNU drug‐resistant glioma cells is significantly lower than that of the parent SWOZ2 cell line. Moreover, down‐regulation of 4E‐BP1 by short interfering RNA significantly impaired the sensitivity of SWOZ2 and U251 cells to carmustine (BCNU). Furthermore, overexpression of 4E‐BP1 with plasmid transfection regained this sensitivity. Clinical studies showed that the expression levels of 4E‐BP1 in primary glioma tissues were markedly higher than those of recrudescent glioma tissues. Taken together, our results suggest that 4E‐BP1 is a novel protein that contributes to acquired drug resistance and it may be a potential target for reversing drug resistance in human glioma.     

2-甲氧基雌二醇对神经胶质瘤荷瘤小鼠的抑瘤作用研究

目的 探讨2-甲氧基雌二醇(2-ME)对神经胶质瘤U251细胞移植瘤的增殖抑制、凋亡及可能的分子机制。方法 构建胶质瘤裸鼠移植瘤模型,经2-ME治疗后分别记录移植瘤的重量、体积; 通过移植瘤HE、免疫组化、TUNEL染色分析2-ME对移植瘤的增殖抑制、凋亡及对Bcl-2、VEGF表达水平的影响; 检测2-ME对裸鼠肝肾功能的影响。结果 2-ME对肿瘤的体积和重量均有抑制作用; HE染色显示实验组出现不同程度的肿瘤细胞密度减低; 免疫组化染色显示,随着2-ME水平的升高,肿瘤组织内Bcl-2、VEGF的表达水平逐渐下降; TUNEL染色显示2-ME诱导裸鼠移植瘤组织细胞凋亡; 肝肾功能检测未见损害发生。结论 2-ME在体内能有效抑制胶质瘤移植瘤的生长、诱导其凋亡,其抗肿瘤作用可能与Bcl-2、VEGF表达水平有关,且无明显毒副作用。     

Epithelial‐to‐mesenchymal transition is involved in BCNU resistance in human glioma cells

Chemotherapy has been considered as an effective treatment for malignant glioma; however, it becomes increasingly ineffective with tumor progression. Epithelial‐to‐mesenchymal transition (EMT) is a process whereby cells acquire morphologic and molecular alterations that facilitate tumor metastasis and progression. Emerging evidence associates chemoresistance with the acquisition of EMT in cancer. However, it is not clear whether this phenomenon is involved in glioma. We used the previously established human glioma cell lines SWOZ1, SWOZ2 and SWOZ2‐BCNU to assess cellular morphology, molecular changes, migration and invasion. We found that BCNU‐resistant cells showed multiple drug resistance and phenotypic changes consistent with EMT, including spindle‐shaped morphology and enhanced pseudopodia formation. Decreased expression of the epithelial adhesion molecule E‐cadherin and increased expression of the mesenchymal marker vimentin were observed in BCNU‐resistant SWOZ1 and SWOZ2‐BCNU cells compared to SWOZ2 cells. Migratory and metastatic potentials were markedly enhanced in SWOZ1 and SWOZ2‐BCNU cells compared to SWOZ2 cells. These data suggest that there is a possible link between drug resistance and EMT induction in glioma cells. Gaining further insight into the mechanisms underlying chemoresistance and EMT may enable the restoration of chemosensitivity or suppression of metastasis.     

Δ-Tetrahydrocannabinol stimulates glucose utilization in C6 glioma cells

The present work was undertaken to study the metabolic response of C6 glioma cells to physiologically relevant doses of Δ⁹-tetrahydrocannabinol (THC), the major active component of marijuana. At those concentrations (i.e. nanomolar range), THC produced a dose-dependent increase in the rates of glucose oxidation to CO₂ and glucose incorporation into phospholipids and glycogen. The THC-induced stimulation of glucose utilization was (i) dose-dependent up to 100 nM THC, (ii) mimicked by the synthetic cannabinoid HU-210, and (iii) prevented by pertussis toxin and the CB1 receptor antagonist SR141716A. In contrast to THC, forskolin markedly depressed CO₂ production, phospholipid synthesis and glycogen synthesis from glucose. The forskolin-induced inhibition of glucose utilization was (i) mimicked by dibutyryl-cAMP, and (ii) prevented by THC, HU-210 and H-7, an inhibitor of the cAMP-dependent protein kinase. Likewise, THC was able to antagonize in part the forskolin-induced elevation of intracellular cAMP concentration, and this antagonistic effect was prevented by SR141716A. However, THC per se did not affect basal cAMP concentration. Results thus indicate that physiologically relevant doses of THC stimulate glucose metabolism in C6 glioma cells through a cannabinoid receptor-mediated process. Although cannabinoid receptors may be coupled to inhibition of adenylyl cyclase in C6 glioma cells, this does not seem to be the mechanism involved in the THC-induced stimulation of glucose metabolism.     

Heparin-binding epidermal growth factor-like growth factor stimulates mitogenic signaling and is highly expressed in human malignant gliomas

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30–40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-α, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms. Received: 18 November 1997 / Revised, accepted: 10 March 1998     

MGMT反义RNA对人脑胶质瘤耐药性的逆转

目的探讨O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)反义RNA对人脑胶质瘤细胞MGMT表达的调节作用。方法将分别为针对MGMTmRNA5’端和全长序列的反义表达载体pLaMT5SN和pLaMTSN,分别导入U251/BCNU耐药细胞,经G418筛选产生稳定抗性克隆aMT5U和aMTU;分别将正义表达载体pLMTSN和空载体pLXSN导入U251/BCNU耐药细胞,经G418筛选产生稳定抗性克隆MTU和XU作为对照。观察转染后的U251/BCNU细胞对MGMT表达的调节作用及U251/BCNU细胞对BCNU敏感性的影响。结果经RT-PCR检测显示,pLaMT5SN和pLaMTSN可在一定程度上降低MGMTmRNA表达水平;MTT检测显示,aMT5U和aMTU细胞对BCNU的敏感性分别较未转染细胞提高7倍和2倍,表明pLaMT5SN和pLaMTSN可在一定程度上提高U251/BCNU细胞对BCNU的敏感性。结论MGMT反义RNA能够在一定程度上抑制脑胶质瘤细胞MGMT的表达水平,提高肿瘤细胞对亚硝基脲类药物的敏感性。     

Regulation of temozolomide resistance in glioma cells via the RIP2/NF-κB/MGMT pathway

BackgroundTemozolomide (TMZ) is a first‐line chemotherapy drug for the treatment of malignant glioma and resistance to it poses a major challenge. Receptor‐interacting protein 2 (RIP2) is associated with the malignant character of cancer cells. However, it remains unclear whether RIP2 is involved in TMZ resistance in glioma.MethodsRIP2 expression was inhibited in TMZ‐resistant glioma cells and normal glioma cells by using small interfering RNA (siRNA) against RIP2. Plasmid transfection method was used to overexpress RIP2. Cell counting kit‐8 assays were performed to evaluate cell viability. Western blotting or immunofluorescence was performed to determine RIP2, NF‐κB, and MGMT expression in cells. Flow cytometry was used to investigate cell apoptosis. TMZ‐resistant glioma xenograft models were established to evaluate the role of the RIP2/NF‐κB/MGMT signaling pathway in drug resistance.ResultsWe observed that RIP2 expression was upregulated in TMZ‐resistant glioma cells, whereas silencing of RIP2 expression enhanced cellular sensitivity to TMZ. Similarly, upon the induction of RIP2 overexpression, glioma cells developed resistance to TMZ. The molecular mechanism underlying the process indicated that RIP2 can activate the NF‐κB signaling pathway and upregulate the expression of O6‐methylguanine‐DNA methyltransferase (MGMT), following which the glioma cells develop drug resistance. In the TMZ‐resistant glioma xenograft model, treatment with JSH‐23 (an NF‐κB inhibitor) and lomeguatrib (an MGMT inhibitor) could enhance the sensitivity of the transplanted tumor to TMZ.ConclusionWe report that the RIP2/NF‐κB/MGMT signaling pathway is involved in the regulation of TMZ resistance. Interference with NF‐κB or MGMT activity could constitute a novel strategy for the treatment of RIP2‐positive TMZ‐resistant glioma.     

胶质瘤干细胞抗凋亡和多重耐药基因的表达

目的 从人脑胶质瘤组织中分离、培养胶质瘤干细胞,并探讨其生物学特性及其抗凋亡和多重耐药基因的表达差异.方法 人脑胶质瘤组织经过原代细胞培养后,用无血清培养方法获得胶质瘤干细胞球,用10%的胎牛血清培养诱导分化,分化前后分别做nestin、tubulin-β、GFAP免疫细胞化学荧光染色,并观察胶质瘤干细胞形态学改变.同时,应用实时荧光定量PCR技术检测livin,livinot,livinβ,survivin,MRPI和MRP3 mRNA的表达.结果 有2例分离培养出干细胞球体,这些球体具有典型的干细胞特性,nestin染色为阳性;在无血清培养基中呈悬浮球样生长,能够自我更新和增殖,在有血清培养基中能够分化,tubulin-β、GFAP染色为阳性.胶质瘤干细胞球livin、livinα、livinβ、survivin和MRP-1 mRNA表达量比较胶质瘤组织表达量都有不同程度的升高,而MRP-3mRNA表达量降低.结论 胶质瘤干细胞抗凋亡和MRP-1基因表达量较胶质瘤表达量有不同程度的升高,提示胶质瘤干细胞比较其同源的胶质瘤细胞具有更强的耐药性,这可能是肿瘤耐药的机制.     

IkappaBalphaM suppresses angiogenesis and tumorigenesis promoted by a constitutively active mutant EGFR in human glioma cells

Human glioma cell lines (G36DeltaEGFR and IN500DeltaEGFR) have been shown to display an enhanced tumorigenic phenotype, when transfected with a constitutively active form of the epidermal growth factor receptor (DeltaEGFR). These cells were transfected with a mutant IkappaBalpha (IkappaBalphaM) that is resistant to phosphorylation and degradation, and hence blocks NF-kappaB activity. Recently, EGFR has been shown to increase the activity of NF-kappaB and to induce angiogenesis. In this report, we asked if IkappaBalphaM gene transfer into human glioma cell lines would inhibit tumorigenicity and angiogenesis in glioma. IkappaBalphaM inhibited in vitro and in vivo expression of vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8). Human glioma xenografts treated with IkappaBalphaM gene transfer exhibited significantly decreased angiogenesis both in an orthotopic and in an ectopic model. The decreased expression of VEGF and IL-8 directly correlated with decreased tumorigenicity, and tumor vascularization. Taken in combination, these results provide strong evidence of IkappaBalphaM's role in regulating glioma angiogenesis even in the presence of constitutive EGFR activation.     

人脑胶质瘤干细胞TGF-32mRNA的表达及意义

目的 检测人脑胶质瘤干细胞(BGSCs)中转化生长因子-β2(TGF-β2) mRNA的表达水平,推断其在胶质瘤免疫治疗中的作用. 方法 选取中国医科大学第一医院神经外科自2008年10月至2009年1月间手术切除的脑胶质瘤标本8例,培养出BGSCs,免疫荧光染色检测肿瘤细胞球CD133、GFAP和TU-20的表达;RT-PCR检测BGSCs中TGF-β2 mRNA的表达,并与对应的原代分化脑胶质瘤细胞进行比较. 结果 肿瘤细胞球CD133呈阳性表达;分化于肿瘤球的细胞可表达GFAP和TU-20; RT-PCR检测发现TGF-β2 mRNA在BGSCs中的表达水平远高于原代分化胶质瘤细胞,差异有统计学意义(t=3.619,P=0.009). 结论 BGSCs中TGF-β2 mRNA呈高表达,这可能是胶质瘤TGF-β2高分泌、产生免疫逃逸并耐受各种常规治疗的决定性因素.     

白藜芦醇对裸鼠U87细胞移植瘤生长及血管生成的影响

目的 研究白藜芦醇(Res)对人脑胶质瘤系U87细胞的体内抗癌活性及血管生成的影响. 方法 BALB/c裸鼠20只,背部皮下接种胶质瘤细胞U87建立皮下移植瘤模型.随机数字表法分为10、100mg/kgRes治疗组,溶剂对照组,空白对照组,每组5只.观察4组裸鼠移植瘤的生长曲线并应用免疫组织化学法检测瘤组织中微血管密度(MVD)及血管内皮生长因子(VEGF)的表达:采用原位末端标记法(TUNEL)检测瘤组织细胞的凋亡. 结果与空白对照组及溶剂对照组相比,100 mg/kg Res治疗组裸鼠移植瘤的体积、重量降低;100 mg/kg Res治疗组瘤组织中MVD、VEGF的表达明显降低,凋亡细胞数增高,差异均有统计学意义(P<0.05). 结论 Res对U87人脑胶质瘤细胞裸鼠移植瘤的生长具有明显抑制作用,这可能与Res导致U87移植瘤细胞凋亡及血管生成减少有关.     

白藜芦醇对裸鼠U87细胞移植瘤生长及血管生成的影响

目的 研究白藜芦醇(Res)对人脑胶质瘤系U87细胞的体内抗癌活性及血管生成的影响. 方法 BALB/c裸鼠20只,背部皮下接种胶质瘤细胞U87建立皮下移植瘤模型.随机数字表法分为10、100mg/kgRes治疗组,溶剂对照组,空白对照组,每组5只.观察4组裸鼠移植瘤的生长曲线并应用免疫组织化学法检测瘤组织中微血管密度(MVD)及血管内皮生长因子(VEGF)的表达:采用原位末端标记法(TUNEL)检测瘤组织细胞的凋亡. 结果与空白对照组及溶剂对照组相比,100 mg/kg Res治疗组裸鼠移植瘤的体积、重量降低;100 mg/kg Res治疗组瘤组织中MVD、VEGF的表达明显降低,凋亡细胞数增高,差异均有统计学意义(P<0.05). 结论 Res对U87人脑胶质瘤细胞裸鼠移植瘤的生长具有明显抑制作用,这可能与Res导致U87移植瘤细胞凋亡及血管生成减少有关.     

脂质体介导CD基因转染SHG-44胶质瘤细胞凋亡的实验研究

目的以含胞嘧啶脱氨酶（CD）基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞，体外观察5-氟胞嘧啶（5-FC）对转染CD基因的胶质瘤细胞的凋亡诱导作用。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因；脂质体Lipofectamine 2000介导pCMVCD质粒转染SHG-44细胞，G418筛选培养获取抗性细胞克隆（即SHG-44／CD细胞）；免疫细胞化学检测SHG-44／CD细胞的CD基因蛋白水平表达；流式细胞仪、TUNEL实验及透射电子显微镜观察5-FC对表达CD基因的SHG-44／CD细胞凋亡的诱导作用。结果含CD基因的pCMVCD质粒成功转染进入SHG-44细胞，获取了含CD基因的SHG-44／CD细胞，免疫细胞化学染色显示SHG-44／CD细胞成功地表达了CD。在含5-FC的培养液中培养，SHG-44／CD细胞出现典型的凋亡形态，TUNEL显示凋亡细胞比例极高；透射电镜可见凋亡改变；流式细胞术检测凋亡率达18．6％，凋亡率呈剂量依赖性。结论建立了SHG-44恶性人脑胶质瘤细胞的CD／5-FC自杀基因系统。诱导SHG-44／CD胶质瘤细胞产生凋亡可能是脑胶质瘤CD基因疗法的重要机制。     

Overexpression of immediate early gene X‐1 (IEX‐1) enhances γ‐radiation‐induced apoptosis of human glioma cell line,U87‐MG

Malignant gliomas are usually incurable even if adjuvant therapy is delivered after neurosurgical treatment. Therefore, to enhance their radiation‐induced apoptosis, it is important to detect the mechanism(s) leading to the death of malignant glioma cells. We report that apoptosis was induced in a time‐dependent manner after γ‐radiation and that irradiated U87‐MG cells (human glioblastoma cell line) expressed immediate early gene X‐1 (IEX‐1) with p53. We also document that their apoptotic sensitivity to γ‐radiation was enhanced by the overexpression of IEX‐1. Our findings suggest that IEX‐1 may represent a new factor for the enhancement of radiation‐induced apoptosis of human glioma cells.     

MGMT反义RNA对人脑胶质瘤耐药性逆转的研究

目的 利用反义技术逆转胶质瘤的耐药性。方法 模拟临床亚硝基脲类药物的用药程序，在体外建立由DNA修复蛋白O^6－甲基鸟嘌呤－DNA甲基转移酶（MGMT）介导的人脑胶质瘤耐药细胞系U251－BCNU，并行细胞药性检测和细胞MGMTmRNA表达的分析，观察MGMT反义RNA对U251/BCNU细胞MGMT表达的调节作用及对U251／BCNU细胞BCNU敏感性的影响。结果 经过反复总共5次的用药过程，首次在体外建立了对BCNU具有稳定抗性的U251／BCNU，其对BCNU的耐受程度约为U251的17倍；RT－PCR结果显示，U251／BCNU细胞有MGMTmRNA的表达。MGMT反义RNA表达载体 LMT5SN和pLaMTSN能够在一定程度上降低U251／BCNU细胞中MGMT mRNA的表达水平，提高其对BCNU的敏感性。结论 MGMT反义RNA能够在一定程度上抑制MGMT的表达水平，提高肿瘤细胞对亚硝基脲类药物的敏感性。