| Project/Area Number |
11671559
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | The University of Tokushima |
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Principal Investigator |
NISHITANI Masa-aki The University of Tokushima, the hospital attached to the medical department, assistant, 医学部・附属病院, 助手 (40304521)
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| Co-Investigator(Kenkyū-buntansha) |
INOUE Yoshio The University of Tokushima, the medical department, assistant professor, 医学部, 助手 (20304511)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | renal cancer / angiogenesis / gene therapy / angiostatin / animal model / マウス / 血管新生抑制治療 |
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| Research Abstract |
1. Detection of angiostatin in conditioned media from stable clones.
The secretion of the angiostatin protein into the cell culture medium were investigated after transient transfection of Renca cells with the two kinds of angiostatin expression constructs. Cells transfected with an expression vector plasmid were used as controls. Cell culture medium and protein extracts were collected 24h after transfection and analyzed by immunoblotting. Western blot analysis with an anti-HA antibody revealed that 58 kDa and 52 kDa expected protein were produced within the cell culture medium. To study the constitutive expression of HA-tagged angiostatin, stable cell clone culture medium were harvested after 4 days incubation. The immunoblots showed the levels of full-length PA angiostatin and COOH-terminus deleted PA angiostatin with molecular masses of 58 and 52 kDa.
2. Angiostatin inhibits murine renal cancer growth in vivo but not in vitro.
Tumors in control mice implanted with mock-transfectant gre
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w rapidly to average volume about 600 mm3. In contrast, significant inhibitions of primary tumor growth was observed in two angiostatin-transfected tumors three weeks after implantation (p<0.05). Five weeks after implantation, the difference was more significant. There was no difference in tumor growth between full-length PA angiostatin and PA deleted angiostatin transfectant. To further investigate whether suppression of tumor growth in the angiostatin-transfected cells was due to an antiangiogenic effect as opposed to direct antitumor activity, each cell growth was tested in vitro. A G418 resistant Renca cell clones that did not express angiostatin, two kinds of clones with high angiostatin expression, and parental Renca cells exhibited the same growth rate. This result indicate that the expression of angiostatin dose not inhibit the growth of Renca cells.
3. Angiostatin inhibits neovasculization of muine renal cancer.
Four weeks after implantation, tumors derived from angiostatin-transfected clones appeared to be small and flat, typical features of tumors with diminished neovascularization. In contrast, large vascularized tumors were observed in the control groups. Histopatholgical findings of H＆E staining showed that the size of blood vessel in tumor tissue obtained from the angiostatin-transfected clones was significantly less than that of controls. Furthermore, immunohistochemical analysis of neo-vascularization by staining with the anti-CD31 antibody revealed that angiostatin suppressed both tumor vessel formation and vascular endothelial cell proliferation. Microvessel density in angiostatin transfected tumors was significantly lower than that of mock-transfected tumors. These results suggest that angiostatin inhibits tumor-induced neovascularization in vivo. Taken together, these data suggest that the mechanism for this significant tumor growth inhibition involves suppression of tumor angiogenesis rather than a primary antitumor effect of angiostatin. Less
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