Non-enhancing de novo glioblastoma: report of two cases

Malignant gliomas arise from two distinct pathways, as de novo lesions or from secondary transformation from low-grade lesions. Herein, we describe the cases of two patients to illustrate the proposition that de novo malignant gliomas can originate as non-enhancing tumors and rapidly progress to a pattern of ring enhancement characteristic of a glioblastoma. Both patients presented with new-onset seizures (simple partial and generalized). Their neurological examinations were unremarkable. Initial MRI evaluations revealed a right precentral gyrus and right medial temporal lobe lesions in each case, respectively. These lesions demonstrated increased T2 signal changes without contrast enhancement. The biopsy of the right frontal lesion in the first patient was consistent with an anaplastic astrocytoma; the second patient was followed expectantly. Repeat MRI for both patients within 17 weeks disclosed ring-enhancing lesions, consistent with an unusually rapid evolution to glioblastoma multiforme (GBM). Subsequent resection of the right medial temporal lesion in the second patient revealed a GBM. Neither tumor displayed abnormal overexpression of P53 by immunohistochemistry. Early MRI of de novo glioblastomas may demonstrate a non-enhancing tumor suggestive of a low-grade lesion. These tumors can rapidly evolve into ring-enhancing lesions more consistent with the traditional imaging findings.     

Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase

The lipid-lowering drugs, statins, induce apoptosis in a variety of tumor cells. Here we investigated the apoptotic effect of the lipophilic statin, simvastatin, in C6 glioma cells and the underlying effects on intracellular signal transduction. Simvastatin inhibited cell proliferation totally after 20 h of treatment as shown by the decrease in proliferating cell nuclear antigen expression in the nucleus. Subsequently, simvastatin caused apoptotic cell death by shrinkage of cytoplasm and condensation of chromatin, and DNA fragmentation. The features of apoptosis were visible only after 48 h of treatment, possibly reflecting a requirement for cell commitment to growth arrest. In immunocytochemical and immunoblotting experiments we have shown that simvastatin markedly increased the phosphorylation of ATF-2 and c-jun in the nucleus of the C6 glioma cells at early time points which was preserved even 24 h after treatment. In contrast, activities of protein kinases Erk1/2 and AKT in the cell survival pathway remained unchanged throughout the treatment. Selective inhibitor of JNK, but not p38 kinase, reduced simvastatin-induced cell death and ATF-2 and c-jun phosphorylation suggesting that JNK-dependent activation of ATF-2 and c-jun may play an important role in simvastatin-induced proliferation inhibition and apoptosis in C6 glioma cells. These observations suggest that statins may have clinical significance in the prevention of glial tumors beyond their cholesterol-lowering effect and JNK may be a rational target for sensitizing glioma cells to chemotherapeutic agents.     

靶向表皮生长因子受体的小分子干扰RNA抑制TJ905人脑胶质瘤细胞增殖和侵袭的实验研究

目的　比较靶向表皮生长因子受体 (EGFR)的小分子干扰RNA与反义RNA构建体对TJ90 5人脑胶质瘤细胞增殖和侵袭的抑制作用。方法　选择人EGFR的二个siRNA靶序列构建靶向EGFR的siRNA表达质粒 ,并以反义EGFRRNA为对照进行脂质体介导的TJ90 5人脑恶性胶质瘤细胞系表达。应用免疫荧光和蛋白印迹检查EGFR的表达水平 ;应用TUNEL法分析细胞凋亡 ,流式细胞术分析细胞周期变化 ,MTT法分析细胞增殖 ;蛋白印迹检测基质金属蛋白酶 9的表达 ,明胶酶谱分析MMP9酶活性 ,Transwell法分析侵袭能力。结果　与TJ90 5细胞和转染空载体的TJ90 5细胞比较 ,转染反义EGFRRNA使EGFR表达下调 82 % ,转染siRNA表达质粒组EGFR表达下调 90 %和 92 %。TJ90 5组和空载转染组几乎没有凋亡细胞 ,反义EGFR转染组与siRNA表达质粒组的凋亡率明显增加 ( χ2 =31 5 4 9,P <0 0 0 1) ;siRNA表达载体转染后S期指数较对照和反义RNA转染组细胞明显减少 ,与TJ90 5组和空载转染组比较 ,转染组细胞自第 1天起存活率均明显下降 (P <0 0 0 1) ,且反义组与siRNA表达载体转染组之间存活率差异有显著意义 (P <0 0 1) ;同时蛋白印迹发现转染siRNA表达载体后MMP 9的表达明显下调 ,明胶酶谱分析发现在反义组与siRNA表达载体转染组MMP 9酶活性明显下降 ,以siRNA     

融合蛋白DT389-hIL-13的克隆表达及其抗胶质瘤作用的初步研究

目的　构建白喉毒素N端 389个氨基酸 (DT389)与人白细胞介素 13(hIL 13)的融合蛋白DT389 hIL 13,并检测其生物学活性。方法　通过聚合酶链反应 (PCR)扩增得到hIL 13的cDNA ,将序列正确的hIL 13及DT389的cDNA片段串联插入表达载体pET30a ,构建表达质粒pET30a/DT389 hIL 13,并对表达产物进行鉴定及初步纯化。该融合蛋白加入培养的U2 51胶质细胞中 ,应用MTS方法检测其对多型性胶质母细胞瘤细胞的杀伤作用。结果　得到序列正确的融合蛋白DT389 hIL 13的表达质粒pET30a/DT389 hIL 13,该质粒在大肠杆菌成功表达 ,经初步纯化后 ,十二烷基磺酸钠 聚丙烯酰胺 (SDS PAGE)凝胶电泳及Western印迹分析表明 ,该融合蛋白对白喉毒素多克隆抗体及hIL 13多克隆抗体都有很好的免疫反应性 ,相对分子质量约为 5 5 0 0 0 ;进一步活性检测发现 ,该融合蛋白对多型性胶质母细胞瘤细胞系U2 5 1的生长有较强的抑制作用 ,且作用存在剂量相关性 ,其半数抑制浓度(IC50 )约为 5× 10 -11mol/L。结论　成功构建了融合蛋白DT389 hIL 13的表达质粒 ,在细胞水平对表达产物进行了初步的活性检测 ,为进一步研制特异性的抗胶质瘤药物打下了基础。     

Expression of Rb2/p130 protein correlates with the degree of malignancy in gliomas

It has been reported that there is an inverse correlation between the immunohistochemical expression of Rb2/p130, a member of the retinoblastoma gene family, and the degree of malignancy in at least some histological types. In order to investigate the expression of this protein in gliomas, we evaluated 58 samples from patients with resected gliomas. We focused on the relationship between the degree of malignancy of the glioma and the immunohistochemical detection of Rb2/p130. Expression of Rb2/p130 was observed in 38 glioma specimens (65.5%), including a high expression level in low-grade glioma specimens (>30% positive cells in 84% of tumors) and a low expression level in high-grade glioma specimens (>30% positive cells in 12% of tumors). The most aggressive of the gliomas exhibited very low to undetectable levels of Rb2/p130. Moreover, we observed an inverse correlation between Rb2/p130 expression and the degree of malignancy. These findings suggest that the differentiation of gliomas might be partially mediated by the Rb2/p130 gene, and that Rb2/p130 expression can additionally be an indicator of a better prognosis in patients with gliomas.     

Clinicopathological examination of glioma by proton magnetic resonance spectroscopy background

Automation of proton magnetic resonance spectroscopy (MRS) in recent years has made it possible for MRS measurement to be performed in a shorter time than before, and the number of reports of its usefulness for the assessment of glioma malignancy has been increasing in the past several years. We studied the efficacy of proton MRS when used for glioma and conducted clinicopathological examination of glioma. The subjects were 15 patients who had received a pathological diagnosis of glioma at our hospital (6 cases of glioblastoma, 1 case of anaplastic astrocytoma, 4 cases of low-grade astrocytoma, and 4 cases of radiation necrosis); Siemens Magnetom Vision 1.5T was used for the study. Regions of interest (ROIs) were defined as the areas where abnormal signals were found on magnetic resonance imaging (MRI). Areas of primary peaks, such as choline (Cho),N-acetylaspartate (NAA), and lactate (Lac), were measured, and the ratios to normal brain tissue were examined. This study revealed a tendency of increased malignancy of glioma with a decrease in NAA. Some cases also displayed a decrease in Cho with an increase in malignancy. Assessment of malignancy must not be based on a single ROI alone, but several ROIs should be assessed comprehensively. Measurement was difficult when the tumor volume was small. Because diagnosis of very early glioma by MRS seemed difficult, other adjunctive diagnoses may be necessary. Proton MRS is very useful for diagnosis of glioblastoma.     

Angiogenesis and antiangiogenic therapy for malignant gliomas

Angiogenesis is crucial to the growth of malignant gliomas. Therefore, antiangiogenic therapy represents a new, promising therapeutic modality for malignant gliomas. This study was designed to define the malignant glioma cases most suitable for antiangiogenic therapy in humans and to demonstrate the efficacy of antiangiogenic therapy in animals. Protein expression of the most potent angiogenic factor, vascular endothelial growth factor (VEGF), and its specific natural inhibitor, soluble Flt-1, as well as vessel architecture, including vessel density, area, and diameter, was evaluated in human malignant glioma samples (24 glioblastomas, 13 anaplastic astrocytomas). Among these, VEGF >1000ng/ml, VEGF/soluble Fltl ratio >1, vessel density >30, and vessel area >7% were prognostic factors for malignant gliomas. Based on these results, we per formed three different antiangiogenic experiments targeted to inhibit VEGF expression in a human malignant glioma (U87) mouse model: anti-VEGF neutralized antibody intraperitoneal injection; interferon-beta intramusclar injection; and transfection of an endogenous nonspecific angiogenesis inhibitor, thrombospondin-1, into glioma cells caused inhibition of VEGF secretion and/or mRNA expression and resulted in glioma growth inhibition of 70%, 84%, and 50%, respectively, compared with control. We conclude that malignant gliomas with high degrees of VEGF expression and vessel areas are good candidates for antiangiogenic therapy, especially that designed to inhibit VEGF expression.     

Improved method for the intracerebral engraftment of tumour cells and intratumoural treatment using a guide screw system in mice

Summary ¶Background. Orthotopic in vivo models for the experimental treatment of malignant gliomas have become indispensable to evaluate the efficacy of novel therapeutic regimens. Recently, a guide screw system was introduced which is implanted into the cranium of small rodents where it facilitates fast, repeated and exactly reproducible intracerebral injections, avoiding time consuming stereotactic procedures. Here we report our experience with this system, and describe several modifications which we introduced to improve its reliability and to simplify its application. Findings. The most important modification made was the optimization of the guide screw implantation site, which needs to be adapted to the age of the mice. Other improvements were the fixation of the guide screw to the cranium of the mice using a two component adhesive. This avoids the tendency, when daily intratumoural injections are performed over several weeks, for the screw to loosen, making precise injections impossible. The injection procedure, initially described as a free-hand approach, was improved by mounting the syringe to a Greenberg retractor. This provided additional stability and speeded up the process. Applying this modified technique, we were able to achieve reproducible results with regard to engraftment rate, tumour growth, and intratumoural treatment in immunocompromized mice. Interpretation. We introduced several major and some minor improvements to make working with the guide screw system more reliable, faster and more comfortable. Daily injections over a period of three weeks using this system are feasible and well tolerated by mice.Published online August 7, 2003     

Glioma after cerebral hydatid disease

Background

The authors present the case of a 31-year-old man with a malignant glioma. He had been treated for cerebral hydatid as a child, and 22 years later he developed a glioma at the site of his previous disease.

Discussion

Could chronic inflammatory change following intracranial hydatid disease have induced neoplastic transformation of glial cells?