Mechanism of inhibitory effect of protein C inhibitor on tumorigenesis and angiogenesis

蛋白C抑制剂（PCI）属于丝氨酸蛋白酶抑制剂（SERPIN）家族，可调节抗凝血蛋白C途径，也可抑制尿纤溶酶原激活物（uPA）， uPA是肿瘤细胞侵袭的介质。PCI可以与肝素结合，并且在肝素存在时，PCI对这些蛋白酶的抑制作用加速。在本研究中，我们评估了人PCI及其无活性衍生物对肿瘤生长、转移和血管生成的影响。重组完整PCI可抑制MDA-231细胞的侵袭性，而活性位点修饰PCI（R354APCI）和蛋白酶裂解PCI n端片段（NTPCI）均不能抑制MDA-231细胞的侵袭性。与表达R354APCI（MDA-R354APCI）或NTPCI（MDA-NTPCI）的MDA-231细胞相比，表达完整PCI（MDA-PCI）的MDA-231细胞的体外侵袭性也显著降低。此外，与MDA-Mock细胞相比，MDA-PCI、MDA-R354APCI和MDA-NTPCI细胞在严重联合免疫缺陷（SCID）小鼠体内的生长和转移潜力显著降低。与含有MDA-Mock细胞的Matrigel植入物相比，含有MDA-PCI、MDA-R354APCI或MDA-NTPCI细胞的血管生成也明显减少。重组完整PCI、R354APCI和NTPCI均能抑制大鼠角膜的体内血管生成和体外血管形成。此外，抗血管生成活性较强，如裂解抗凝血酶和色素上皮衍生因子。随后，我们使用Western- blot分析检测了PCI在肝素存在或不存在的情况下对血管内皮生长因子（VEGF）诱导的丝裂原活化蛋白激酶（MAPK）激活的影响，结果表明，只有在肝素存在的情况下，PCI才会抑制MAPK的激活。总体而言，本研究表明PCI通过抑制MAPK活化而独立于蛋白酶抑制活性抑制血管生成，PCI上的肝素结合位点是发挥其抗血管生成活性的重要途径

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