| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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