Increased levels of tissue endostatin in human malignant gliomas.

PURPOSE: Malignant gliomas are typically angiogenic and express greater amounts of angiogenic factors. We examined glioma tissues for their expression of an endogenous inhibitor of angiogenesis, endostatin, a COOH-terminal fragment of collagen XVIII. EXPERIMENTAL DESIGN: We examined frozen tissues from 51 patients with astrocytic tumors (grade 2, 13; grade 3, 9; and grade 4, 29). Frozen tissues were subjected to immunoblot analysis and immunohistochemistry for endostatin. Tumor vascular density was determined by calculating the percentage of tumor capillary vessel areas/tissue section area. Tissue concentrations of vascular endothelial growth factor and basic fibroblast growth factor were examined by enzyme immunoassay. RESULTS: The levels of endostatin protein estimated by immunoblotting were significantly higher in grade 4 than lower-grade glioma tissues. The immunoreactive bands for endostatin were identified as the fragment derived from noncollagenous domain 1 of collagen XVIII, a peptide 15 residues longer than endostatin toward the NH(2)-terminal end, by NH(2)-terminal amino acid sequencing. In addition to an intense immunoreactivity for endostatin in tumor blood vessels, sections from malignant gliomas showed widely distributed immunoreactivity around tumor cells near the hyperplastic microvessels. The tumor vascular density and the levels of vascular endothelial growth factor in grade 4 glioma tissues were significantly higher than grade 2 and grade 3 gliomas, whereas the levels of basic fibroblast growth factor were the same. CONCLUSIONS: The results indicate a positive correlation between the levels of tissue endostatin and malignancy grades in gliomas. The endostatin may be released near the tumor blood vessels with hyperplasia to counteract angiogenic stimuli in malignant gliomas.     

Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas

Malignant gliomas maintain a poor prognosis and survival rate due to their marked local invasive growth and neovascularization. Matrix metalloproteinases (MMPs) have been implicated in glioma invasion and angiogenesis, but it is unknown whether they are produced by the tumor cells or surrounding stroma. Using in situ hybridization and immunohistochemistry, we found expression of mRNA for both gelatinase-A (MMP2) and gelatinase-B (MMP9) localized to tumor cells and vascular structures in glioma sections. Gelatinase-A protein expression was detected most prominently in tumor cells, with very little signal seen in vasculature. Gelatinase-B protein expression was prominent in vascular structures but was also expressed in tumor cells. Our data show that these proteases are produced by glioma cells and vascular structures and suggest that synthetic MMP inhibitors might be useful in this disease.     

Epigenetics in human gliomas

Aberrant epigenetic landscapes and their involvement in genesis and progression of tumors, as well as in treatment responses and prognosis, indicate one of the most emerging fields in cancer research. In gliomas, the most common human primary brain tumors, and in particular in glioblastoma, the most malignant and devastating brain tumor entity in adults, the elucidation of distinct patterns of aberrant DNA methylation, histone modification, and miRNA expression and their interrelationship has fundamentally changed our point of view on these highly heterogeneous tumors. In the current review article, we address the basic principles of epigenetic control in gliomas, their current and putative future role in prognostic and predictive models and possible interactions within the epigenetic network. We discuss diagnostic and therapeutic opportunities appearing at horizon of epigenetic research. Moreover, we present current and propose future clinical workflow models for molecular characterization of malignant gliomas.     

重组人血管内皮细胞抑制因子在大鼠体内的组织分布及药代动力学

目的：研究重组人血管内皮细胞抑制因子（rhEndostatin）静脉注射后在Wistar大鼠体内的药代动力学过程，为其临床应用提供药代动力学数据。方法：应用放射性核素示踪技术结合三氯醋酸沉淀法（TCA沉淀法），对^125I-rhEndostatin静脉注射大鼠后不同时间、不同组织内的核素分布量进行测定，并将血药浓度-时间数据经计算机拟合，计算出相应参数。结果：^125I-rhEndostatin静脉注射大鼠后，药物的分布半衰期平均为（0．154±0．024）h，消除半衰期为（4.03±0．58）h。血药浓度-时间曲线下面积（AUC）与剂量呈正相关，相关系数为0．9940。血浆清除率（CLs）均值为（0．165±0．024）L／h，高、中、低剂量CLs基本相同。该药在大鼠肝、肺、脾、胃、甲状腺中有较高聚集，主要经肾脏排泄。结论：^125I-rhEndostatin在大鼠体内的药代动学过程基本符合线性药动学特征，血药浓度-时间曲线符合二房室模型。     

重组人血管内皮抑制素临床应用进展

重组人血管内皮抑制素具有抗血管生成作用,副作用小。与化疗联合或单药治疗晚期恶性肿瘤,可提高疗效,改善生活质量,延长生存时间。在临床具有一定的应用前景。     

Glycosphingolipids of human gliomas

Histologically characterized human gliomas of various grades of malignancy obtained during surgery were extracted, and their glycolipids were isolated and partially identified. Among the gliomas analyzed, three types of glycolipid component distribution could be identified. The glycosphingolipid (GSL) type I pattern correlated closely with that of the most malignant gliomas (Grade IV). Its neutral GSLs consisted of glucosyl- and, as a major component, dihexosylceramide, in addition to globo- and neolactotetraosylceramide. Galactosylceramide and sulfatide were absent. The gangliosides of GSL type I were almost exclusively of the GLac family, aside from small amounts of neolacto-series-derived species. The neutral components of GSL type II were similar to those of GSL type I. The acidic compounds of GSL type II were gangliosides of the Gtri family and trace amounts of neolacto-series sialoglycolipids, in addition to GLac1 and GLac2. GSL type II contained no Gtet gangliosides and no sulfatide. The GSL type III pattern was that of the most benign gliomas, with all glycolipids present that are found in normal brain and, in addition, those of the GSL type II.     

Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models

Oncolytic virotherapy is a potential treatment modality under investigation for various malignancies including malignant brain tumors. Unlike some other natural or modified viruses that show oncolytic activity against cerebral neoplasms, the rodent parvovirus H-1 (H-1PV) is completely apathogenic in humans. H-1PV efficiently kills a number of tumor cells without harm to corresponding normal ones. In this study, the concept of H-1PV-based virotherapy of glioma was tested for rat (RG-2 cell-derived) and for human (U87 cell-derived) gliomas in immunocompetent and immunodeficient rat models, respectively. Large orthotopic rat and human glioma cell-derived tumors were treated with either single stereotactic intratumoral or multiple intravenous (iv) H-1PV injections. Oncolysis was monitored by magnetic resonance imaging and proven by histology. Virus distribution and replication were determined in brain and organs. In immunocompetent rats bearing RG-2-derived tumors, a single stereotactic intratumoral injection of H-1PV and multiple systemic (iv) applications of the virus were sufficient for remission of advanced and even symptomatic intracranial gliomas without damaging normal brain tissue or other organs. H-1PV therapy resulted in significantly improved survival (Kaplan–Meier analysis) in both the rat and human glioma models. Virus replication in tumors indicated a contribution of secondary infection by progeny virus to the efficiency of oncolysis. Virus replication was restricted to tumors, although H-1PV DNA could be detected transiently in adjacent or remote normal brain tissue and in noncerebral tissues. The results presented here and the innocuousness of H-1PV for humans argue for the use of H-1PV as a powerful means to perform oncolytic therapy of malignant gliomas.     

Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype.

Allelic alterations of chromosomes 1 and 19 are frequent events in human diffuse gliomas and have recently proven to be strong predictors of chemotherapeutic response and prolonged survival in oligodendrogliomas (Cairncross et al., 1998; Smith et al., submitted). Using 115 human diffuse gliomas, we localized regions of common allelic loss on chromosomes 1 and 19 and assessed the association of these deletion intervals with glioma histological subtypes. Further, we evaluated the capacity of multiple modalities to detect these alterations, including loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The correlation coefficients for detection of 1p and 19q alterations, respectively, between modalities were: 0.98 and 0.87 for LOH and FISH, 0.79 and 0.60 for LOH and CGH, and 0.79 and 0.53 for FISH and CGH. Minimal deletion regions were defined on 19q13.3 (D19S412-D19S596) and 1p (D1S468-D1S1612). Loss of the 1p36 region was found in 18% of astrocytomas (10/55) and in 73% (24/33) of oligodendrogliomas (P < 0.0001), and loss of the 19q13.3 region was found in 38% (21/55) of astrocytomas and 73% (24/33) of oligodendrogliomas (P = 0.0017). Loss of both regions was found in 11% (6/55) of astrocytomas and in 64% (21/33) of oligodendrogliomas (P < 0.0001). All gliomas with LOH on either 1p or 19q demonstrated loss of the corresponding FISH probe, 1p36 or 19q13.3, suggesting not only locations of putative tumor suppressor genes, but also a simple assay for assessment of 1p and 19q alterations as diagnostic and prognostic markers.     

重组人血管内皮抑素对血管新生的影响研究

目的 探讨重组人血管内皮抑素（Endostar,恩度）对血管内皮细胞趋化、迁移、粘附、增殖及小管形成等与血管新生相关生物学行为的影响。方法 以原代培养的人脐静脉内皮细胞（HUVEC）为细胞模型,通过Boyden小室荧光定量分析、划痕试验、HUVEC荧光定量粘附分析、CFSE染色流式细胞术、CCK-8定量检测、小管形成试验和Matrigel栓试验研究恩度对HUVEC与血管新生相关的生物学行为的影响。结果 恩度在5～50 000ng／ml范围内可抑制血管内皮生长因子诱导HUVEC的迁移运动,且浓度为50ng／ml和500ng／ml时效果最明显;恩度在5～50 000ng／ml间可呈剂量依赖的方式抑制HUVEC向损伤部位的迁移。与0ng／ml相比,50、500和5000ng／ml 恩度处理的HUVEC的粘附率、增殖率及HUVEC形成网状小管结构的数量、面积和长度均降低,差异均有统计学意义（P＜0.05）; Matrigel栓实验结果显示,恩度在50～5000ng／ml间对SCID小鼠体内血管新生有明显的抑制作用。结论 恩度在细胞水平能抑制HUVEC与血管新生相关的生物学行为,包括HUVEC的趋化、迁移、粘附、增殖和小管形成;在动物水平能抑制SCID小鼠体内的血管新生,据此推断恩度能抑制血管新生。     

14例脑胶质瘤染色体分析

目的：采用简便的脑肿瘤染色体制备方法，分析胶质瘤染色体异常在肿瘤发生中的意义。方法：本文用直接制片法和短期培养法，对１４例人脑胶质瘤新鲜瘤组织进行细胞遗传学研究。结果：１４例瘤组织１２例收获到分裂相，其中组织分级较低的８例干系核型正常，仅少数细胞染色体畸变；４例恶性度较高的肿瘤干系核型异常。常见的染色体异常有Ｙ、２２、８、１７号染色体丢失，１、２、３、４、和２２号结构重排。两例胶质瘤发现１７号染色体丢失，在以往文献中罕见报道。结论：胶质瘤中染色体异常随病理诊断恶性度升高而增多，本研究中的非随机染色体畸变可能与胶质瘤病因学相关。     

Molecular biology of human gliomas

Human gliomas are the most common primary central nervous system neoplasm, and they are a complex, heterogeneous, and difficult disease to treat. In the past two decades, advances in molecular biology have revolutionized our understanding of the mechanism by which these neoplasms are initiated and progress. While surgery, radiation therapy, and chemotherapy have roles to play in the treatment of patients with gliomas; these therapies are self-limited because of the intrinsic resistance of glioma cells to therapy, and the diffusely infiltrating nature of the lesions. It is now known that malignant gliomas arise from a number of well-characterized genetic alterations and activations of oncogenes and inactivation of tumor suppressor genes. These genetic alterations disrupt critical cell cycle, growth factor activation, apoptotic, cell motility, and invasion pathways that lead to phenotypic changes and neoplastic transformation. Research in each of these fields has uncovered potential therapeutic targets that look promising for disease control. Gliomas can now be modeled with fidelity and reproducibility using several transgenic and knockout strategies. Transgenic mouse models are facilitating the testing of various therapeutic strategies in vivo. Finally, the recognition of the putative brain tumor stem cell, the tumor initiating cell in brain cancer, provides an enticing target through which we could eliminate the source of the brain tumor with increased efficacy and less toxicity to normal tissues. In this review, we provide an up-to-date discussion of the many of key technologies and tools that are being used in molecular biology to advance our understanding of the biological behavior of human malignant gliomas.     

Activity of lysosomal exoglycosidases in human gliomas

There is a lot of data suggesting that modifications of cell glycoconjugates may be important in progression of cancer. In the present work we studied activities of lysosomal exoglycosidases: β-hexosaminidase and its isoenzymes A and B, β-galactosidase and α-mannosidase, in human gliomas. Enzyme activity was determined spectrophotometrically based on the release of p-nitrophenol from p-nitrophenyl-derivative of appropriate sugars. The activities of the exoglycosidases tested were significantly higher in malignant glial tumors than in control tissue (normal brain tissue) and non-glial tumors. The highest activities of exoglycosidases were observed in high-grade gliomas, and a positive correlation of enzyme activities and degree of malignancy was noted. Our results suggest that lysosomal exoglycosidases may participate in the progression and dynamical development of glial tumors.     

Absence of methylthioadenosine phosphorylase in human gliomas

All normal mammalian tissues contain methylthioadenosine phosphorylase, which plays a role in the recycling of purines and methionine consumed during polyamine synthesis. A complete deficiency of methylthioadenosine phosphorylase has been reported in some human leukemias and lymphomas and in a few solid tumors. The exact incidence of the enzyme deficiency among fresh human tumor specimens has been difficult to establish because the measurement of enzyme catalytic activity is laborious and requires carefully preserved specimens. We have generated two antibodies against methylthioadenosine phosphorylase and have used them to develop a simple immunoblot assay for the enzyme. Specifically, studies showed that all cells with catalytically active methylthioadenosine phosphorylase had a 32-kDa band that reacted with the anti-enzyme antibodies. In a reciprocal manner, all malignant cell lines that were naturally deficient in methylthioadenosine phosphorylase activity lacked detectable immunoreactive enzyme protein. The immunoassay was used to analyze human gliomas. Seventy-five % (9 of 12) of the gliomas were completely methylthioadenosine phosphorylase deficient. This common metabolic difference between most gliomas and all normal cells is a potential target for tumor-specific chemotherapy.     

重组人内皮抑素对肺鳞癌放射增敏作用的体外实验研究

目的 探讨国产重组人内皮抑素是否对肺鳞癌细胞系H-520具有放射增敏作用.方法 取指数生长期的人肺鳞癌细胞系H-520细胞,用成克隆实验检测内皮抑素的细胞毒性和各实验组的克隆形成能力,计算各组细胞存活分数,利用多靶单击模型拟合细胞存活曲线.流式细胞术检测细胞凋亡、细胞周期分布变化及活化的Caspase蛋白片段水平.结果 成克隆实验中内皮抑素联合照射组较照射组D0、Dq、D10、SF2值均低,放射增敏比为1.50(D0值之比).流式细胞术检测细胞凋亡示内皮抑素+照射组细胞凋亡率分别为22.38%±1.61%、35.01%±1.16%、46.83%±2.06%、64.08%±4.28%,照射组分别为4.27%±0.29%、14.3%±1.15%、28.49%±1.58%、54.79%±1.89%,两组凋亡率差异有统计学意义(t=19.17、17.79、25.64、3.44,P值均<0.05).内皮抑素可诱导H-520细胞G0+G1阻滞,而照射则诱导G2+M期阻滞.内皮抑素+照射组活化Caspase蛋白片段表达增加(62.7%±1.9%)较对照组(12.1%±0.1%)、内皮抑素组(54.6%±1.0%)和照射组(34.1%±1.2%)显著增高(t=46.69、6.55、22.54,P值均<0.05;).结论 内皮抑素可通过抑制H-520细胞牛长、促进凋亡、细胞周期重分布来达到放射增敏作用.     

重组人内皮抑素30肽的抗肿瘤作用研究

目的:探讨重组人内皮抑素30肽对肝癌细胞的生物活性及其可能的作用途径.方法:建立HepG2肝癌细胞的单层细胞和多细胞球模型,采用MTT方法和流式细胞技术检测重组人内皮抑素30肽对单层细胞和多细胞球的生长抑制率和细胞调亡率;采用免疫组化和图像分析技术了解重组人内皮抑素30肽与肝癌细胞表面整合素结合的情况.结果:MTT实验表明,在二维细胞中,重组人内皮抑素30肽对HepG-2肝癌细胞表现出明显的抑制作用.在单层细胞和多细胞球中,重组人内皮抑素30肽均可导致细胞凋亡.免疫组化结果显示,无论在单层细胞还是多细胞球中,使用重组人内皮抑素30肽组HepG-2肝癌细胞表面的α5β1整合素和αVβ3整合素的表达均比对照组明显降低(P<0.05).结论:重组人内皮抑素30肽对HepG2肝癌细胞具有直接的杀伤作用.重组人内皮抑素30肽可与HepG-2肝癌细胞表面的α5β1整合素和αVβ3整合素结合发挥其抗肿瘤作用.     

脑胶质瘤旋转容积调强剂量学研究

目的:研究旋转容积调强(RapidArc)在脑胶质瘤中应用的适用性和剂量学特性。方法:对10例脑胶质瘤患者分别设计RapidArc和固定野逆向调强放射治疗(dIMRT)计划。利用剂量体积直方图比较两种计划中靶区、危及器官的剂量学差异,并比较两种治疗计划的总机器跳数(MU)和治疗时间。结果:与dIMRT相比,RapidArc的CTV1平均剂量Dmean,CTV2平均剂量Dmean、最小剂量Dmin稍高,差异有统计学意义(P=0.017,0.036,0.002);CTV2的适合度指数CI值更接近于1(P=0.007);RapidArc中危及器官各指标之间仅有患侧视神经Dmax稍高(P=0.003);其余各指标间差异无统计学意义(P>0.05)。机器跳数RapidArc平均减少了40.9%,治疗时间约是dIMRT的1/3。结论:在脑胶质瘤中,RapidArc计划可以达到或优于dIMRT计划的剂量分布,满足临床剂量学要求,并且具有较少总MU、总治疗时间的优势。     

Overexpression and amplification of STK15 in human gliomas

The serine/threonine kinase 15 (STK15) at chromosome 20q13.2 is frequently shown to be amplified and overexpressed in several human cancers. STK15 has been reported to act as a cell cycle regulator and its overexpression induces centrosome amplification and aneuploidy. Recently we showed that STK15 even plays a role in human malignant brain tumours and we described an amplification of the gene in 31% of the investigated gliomas. In this study we scrutinized the correlation of increased STK15 on DNA and mRNA levels in gliomas of different histological grades. Southern blotting confirmed the amplification frequency of the STK15 gene, which had been previously detected by comparative PCR. In total, DNA gains were found in 26% of the investigated gliomas. Interestingly, we detected overexpression of STK15 mRNA in 60% of the analyzed brain tumours. The elevated expression does not strongly correlate with gains on DNA level, but all cases with an amplification of the STK15 gene display overexpression. Gains of the STK15 gene seem to occur irrespectively of the histological grades of the tumours, so that STK15 probably is not a progression associated factor. Amplification and overexpression of the kinase rather represent a primary alteration in human gliomas, which could play an important role as an early event in all glioma subtypes.