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Development of new treatment fr malignant astrocytoma using antiproteolytic activities.

利用抗蛋白水解活性开发恶性星形细胞瘤新疗法。

基本信息

批准号：
15591557
负责人：
YAMAMOTO Masaaki
金额：
$ 2.24万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591557/
关键词：
glioblastoma Invasion diphtheria toxin A chain Receptor associated protein U251MG gliomas cell line Nuclear Factor Kappa-B pentoxyfilin MMP inhibitor ジフテリアトキシン A鎖

项目摘要

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

纯化了重组白喉毒素，该毒素在片段B中具有两个点突变，可阻断与哺乳动物细胞的结合。这种重组白喉毒素具有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中，重组白喉毒素显示出抗肿瘤活性。将U251 MG细胞接种于小鼠脑内1周后，瘤内直接注射重组白喉毒素，重组白喉毒素治疗组颅内实验胶质瘤体积缩小。为了将蛋白毒素靶向恶性星形细胞瘤细胞，我们现在正在开发重组白喉毒素和受体相关蛋白的组合蛋白，其特异性结合细胞表面脂蛋白受体蛋白（LRP）。与正常胶质细胞相比，LRP在胶质瘤细胞中特异性表达。我们还研究了Pentoxyfilin的抗肿瘤活性， ...更多信息抑制核κ B（NF-κ B）活性。在小鼠实验性颅内胶质瘤模型中，Pentoxyfilin处理的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活性的降低可能是NF-κ B活性降低的结果，NF-κ B控制MMP-9的表达。我们还研究了乙酰肝素酶内切糖苷酶在人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乙酰肝素酶的作用之间的关系。乙酰肝素酶mRNA在胶质母细胞瘤中几乎检测不到，而在转移性脑肿瘤中通过逆转录-聚合酶链反应（RT-PCR）检测到。石蜡包埋切片中乙酰肝素酶的免疫组化显示，转移性脑肿瘤中的肿瘤细胞，特别是侵入血管的肿瘤细胞，表现出强烈的乙酰肝素酶免疫反应性。乙酰肝素酶存在于两个高侵袭性胶质瘤细胞系U87 MG和U251 MG中，其在小鼠实验肺转移模型中没有测定的转移能力。这些细胞系中的乙酰肝素酶活性与高转移性黑素瘤细胞系Bib-F1中的乙酰肝素酶活性几乎相同。然而，接种U87 MG细胞后，免疫组化分析在体内皮下或脑内实验性胶质瘤中不再检测到乙酰肝素酶。实时荧光定量PCR结果显示，U87 MG体外培养胶质瘤细胞中乙酰肝素酶mRNA的表达量是裸鼠体内U87 MG胶质瘤组织中乙酰肝素酶mRNA表达量的10倍。这些结果表明，乙酰肝素酶的表达下调胶质母细胞瘤在体内，很少转移到中枢神经系统以外的远处器官。乙酰肝素酶与胶质母细胞瘤对周围正常脑组织的侵袭性无关。少