Development of new treatment fr malignant astrocytoma using antiproteolytic activities.

纯化了一种重组白喉毒素，该毒素在片段B中有两个点突变，阻止了与哺乳动物细胞的结合。该重组白喉毒素具有A片段活性，可抑制哺乳动物细胞中蛋白质的合成。在初步的实验性小鼠脑胶质瘤模型中，该重组白喉毒素具有抗肿瘤活性。小鼠脑内接种U251 MG细胞1周后，直接瘤内注射重组白喉毒素，重组白喉毒素治疗组脑内胶质瘤体积缩小。为了将蛋白毒素靶向恶性星形细胞瘤细胞，我们目前正在开发重组白喉毒素和受体相关蛋白的联合蛋白，这种蛋白可以与细胞表面脂蛋白受体蛋白(LRP)特异性结合。与正常神经胶质细胞相比，LRP在胶质瘤细胞中特异表达。我们还研究了…的抗肿瘤活性。More抑制核转录因子kappaB(NKB)的活性。在小鼠实验性脑胶质瘤模型中，戊氧非林治疗组小鼠颅内肿瘤体积缩小，这与基质金属蛋白酶-9(MMP9)活性表达降低有关。MM P-9活性降低可能与其抑制基质金属蛋白酶-9表达的核因子-kB活性降低有关。我们还研究了内切糖苷酶乙酰肝素酶在人脑胶质母细胞瘤和脑转移瘤以及正常脑组织中的表达，以探讨其与胶质母细胞瘤生物学特性之间的关系。在体内的胶质母细胞瘤中几乎检测不到乙酰肝素酶的mRNA，而在脑转移瘤中通过逆转录聚合酶链式反应(RT-PCR)检测到该基因的表达。石蜡包埋切片肝素酶免疫组织化学显示，脑转移瘤中的肿瘤细胞，尤其是侵犯血管的肿瘤细胞呈强的肝素酶免疫反应。体外培养的两种高侵袭性胶质瘤细胞株U87 MG和U251 MG中均存在乙酰肝素酶，而小鼠实验性肺转移模型不能检测到这两种细胞的转移能力。这些细胞系的乙酰肝素酶活性与高转移性黑色素瘤细胞系Bib-F1几乎相同。然而，裸鼠接种U87 MG细胞后，免疫组织化学分析在皮下或脑内实验性胶质瘤中不再检测到乙酰肝素酶。实时定量聚合酶链式反应显示，体外培养的U87 MG人脑胶质瘤细胞中乙酰肝素酶基因的表达水平是裸鼠体内U87 MG人脑胶质瘤组织的10倍。这些结果表明，肝素酶在体内胶质母细胞瘤中的表达下调，这种肿瘤很少转移到中枢神经系统以外的远处器官。肝素酶与体内胶质母细胞瘤对周围正常脑组织的侵袭性无关。较少

A recombinant diphtheria toxin, which has two point mutation in fragment B that blocks binding to mammalian cells, was purified. This recombinant diphtheria toxin has A fragment activity, which inhibits protein synthesis in mammalian cells. In preliminary experimental mouse intracranial gliomas model, this recombinant diphtheria toxin showed anti-tumor activity. One week after inoculation of the U251MG cell in mouse brain, recombinant diphtheria toxin was injected in tumor directly The intracranial experiment gliomas showed decreased tumor volume in the recombinant diphtheria toxin treatment group. To target protein toxin to malignant astrocytoma cells, we are now developing combined protein with recombinant diphtheria toxin and receptor-associated protein, which specifically binds to the cell surface lipoprotein receptor protein (LRP). LRP was specifically expressed in gliomas cells compared with normal glial cells. We also investigated the anti-tumor activity of Pentoxyfilin, which i … More nhibits the activities of nuclear kappa-B (NF-kB). In mouse experimental intracranial gliomas model, the volume of intracranial tumor was decreased in Pentoxyfilin treated mice, which was associated with decreased expression of matrix metalloprotease-9 (MMP-9) activity. Decreased activity of MM P-9 might be consequence of decreased activity of NF-kB, which control the expression of MMP-9. We also investigated the presence of endoglycosidase heparanase in human glioblastoma and metastatic brain tumors as well as in normal brain tissue to explore the relationship between biological characteristics of glioblastoma and the role of heparanase. Heparanase mRNA was almost undetectable in glioblastoma in vivo, while it was frequently seen in metastatic brain tumors by reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry of heparanase in paraffin-embedded sections showed that neoplastic cells in metastatic brain tumors, especially in tumor cells that invaded blood vessels, exhibited intense heparanase immunoreactivity. Heparanase was present in two highly invasive glioma cell lines U87MG and U251MG In vitro, which did not have metastatic capability assayed by an experimental pulmonary metastases model in mice. The activity of heparanase in these cell lines was almost the same as that in the highly metastatic melanoma cell line Bib-F1. However, after nude mice were inoculated with U87MG cells, heparanase was no longer detected in subcutaneous or intracerebral experimental glioma in vivo by immunohistochemical analysis. By real-time quantitative PCR, there was a 10-fold increase in heparanase mRNA in U87MG glioma cells in vitro compared to experimental glioma tissue of U87MG in vivo in nude mice. These results indicate that the expression of heparanase was down-regulated in glioblastoma in vivo, which rarely metastasizes to distant organs outside the central nervous system. Heparanase is not implicated in the invasiveness of glioblastoma to surrounding normal brain tissue in vivo. Less