|Budget Amount *help
¥3,980,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
Protein C inhibitor(PCI), which belongs to serine protease inhibitor(SERPIN) family, regulates the anticoagulant protein C pathway and also inhibits urinary plasminogen activator(uPA), a mediator of tumor cell invasion. PCI can bind to heparin, and inhibition of these proteases by PCI is accelerated in the presence of heparin. In the present study, we evaluated the effect of human PCI and its inactive derivatives on tumor growth, metastasis and angiogenesis. The invasiveness of MDA-231 cells was inhibited by recombinant intact PCI, but not by reactive site-modified PCI(R354APCI) or by the N-terminal fragment of protease-cleaved PCI(NTPCI). The in vitro invasiveness of MDA-231 cells expressing intact PCI(MDA-PCI) was also significantly decreased as compared to MDA-231 cells expressing R354APCI(MDA-R354APCI) or NTPCI(MDA-NTPCI). Further, in vivo growth and metastatic potential of MDA-PCI, MDA-R354APCI and MDA-NTPCI cells in severe combined immunodeficient(SCID) mice were significantly decreased as compared to MDA-Mock cells. Angiogenesis was also significantly decreased in Matrigel implant containing MDA-PCI, MDA-R354APCI or MDA-NTPCI cells as compared to that containing MDA-Mock cells. In vivo angiogenesis in rat cornea and in vitro tube formation were also inhibited by recombinant intact PCI, R354APCI and NTPCI. Furthermore, the anti-angiogenic, activity was strong as cleaved antithrombin and pigment epithelium-derived factor. Subsequently, we examined the effect of PCI on vascular endothelial growth factor(VEGF) -induced activation of mitogen-activated protein kinase(MAPK) in the presence or absence of heparin using Western- blot analysis, and suggests that MAPK activation is inhibited by PCI only in the presence of heparin.
Overall, this study showed that, PCI inhibits angiogenesis independently of its protease inhibitory activity by inhibiting MAPK activation, and heparin-binding site on PCI is important for exerting its anti-angiogenic activity