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中具有两点突变，可以阻止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的蛋白质合成。在小鼠颅内胶质瘤模型的初步实验中，该重组白喉毒素显示出抗肿瘤活性。小鼠脑内U251MG细胞接种1周后，直接在肿瘤内注射重组白喉毒素，重组白喉毒素治疗组颅内实验胶质瘤肿瘤体积减小。为了将蛋白毒素靶向于恶性星形细胞瘤细胞，我们正在开发重组白喉毒素和受体相关蛋白的联合蛋白，该蛋白与细胞表面脂蛋白受体蛋白（LRP）特异性结合。与正常胶质细胞相比，LRP在胶质瘤细胞中特异性表达。此外，我们还研究了Pentoxyfilin的抗肿瘤活性，它可以抑制细胞核κ b （NF-kB）的活性。在小鼠实验性颅内胶质瘤模型中，己氧菲林治疗小鼠颅内肿瘤体积减小，这与基质金属蛋白酶-9 （MMP-9）活性表达降低有关。MMP-9活性降低可能是NF-kB活性降低的结果，NF-kB控制着MMP-9的表达。我们还研究了内糖苷酶肝素酶在人类胶质母细胞瘤和转移性脑肿瘤以及正常脑组织中的存在，以探讨胶质母细胞瘤的生物学特性与肝素酶的作用之间的关系。肝素酶mRNA在体内胶质母细胞瘤中几乎检测不到，而通过逆转录聚合酶链反应（RT-PCR）在转移性脑肿瘤中却很常见。肝素酶免疫组化石蜡包埋切片显示，转移性脑肿瘤的肿瘤细胞，尤其是侵袭血管的肿瘤细胞表现出强烈的肝素酶免疫反应性。肝素酶存在于两种高侵袭性胶质瘤细胞系U87MG和U251MG中，通过小鼠肺转移模型检测其不具有转移能力。肝素酶在这些细胞系中的活性与高度转移的黑色素瘤细胞系Bib-F1中的活性几乎相同。然而，裸鼠接种U87MG细胞后，免疫组化分析在体内皮下和脑内实验性胶质瘤中均未检测到肝素酶。通过实时定量PCR检测，体外U87MG胶质瘤细胞中heparanase mRNA的表达量比裸鼠体内U87MG胶质瘤组织中heparanase mRNA的表达量增加了10倍。这些结果表明，肝素酶在胶质母细胞瘤体内表达下调，很少转移到中枢神经系统以外的远端器官。肝素酶在体内与胶质母细胞瘤对周围正常脑组织的侵袭性无关。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