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

蛋白C抑制物(PCI)属于丝氨酸蛋白酶抑制物(SERPIN)家族，调节抗凝蛋白C途径，也抑制肿瘤细胞侵袭的介质尿纤溶酶原激活物(UPA)。PCI能与肝素结合，在肝素存在下，对这些酶的抑制作用被加速。在本研究中，我们评价了人经皮冠状动脉介入治疗及其非活性衍生物对肿瘤生长、转移和血管生成的影响。重组完整的pCI可抑制MDA-231细胞的侵袭力，但不能被反应性位点修饰的pCI(R354APCI)或蛋白酶裂解的pCI的N端片段(NTpCI)所抑制。与表达R354APCI的MDA-231细胞(MDA-R354APCI)或表达NTPCI的细胞(MDA-NTPCI)相比，表达完整PCI的MDA-231细胞的体外侵袭力显著降低。此外，与MDA-Mock细胞相比，严重联合免疫缺陷(SCID)小鼠体内MDA-PCI、MDA-R354APCI和MDA-NTPCI细胞的生长和转移能力显著降低。与含有MDA-Mock细胞的Matrigel种植体相比，含有MDA-PCI、MDA-R354APCI或MDA-NTPCI细胞的Matrigel植入物的血管生成也显著减少。重组完整pCI、R354ApCI和NTpCI也抑制了大鼠角膜的体内血管生成和体外新生血管的形成。此外，其作为裂解的抗凝血酶和色素上皮源性因子具有较强的抗血管生成活性。随后，我们通过Western-印迹分析检测了在有无肝素存在的情况下，经皮冠状动脉介入治疗对血管内皮细胞生长因子诱导的丝裂原活化蛋白激酶(MAPK)的激活的影响，提示只有在有肝素存在的情况下，经皮冠状动脉介入治疗才能抑制MAPK的激活。综上所述，本研究表明，经皮冠状动脉介入治疗通过抑制MAPK的激活而不依赖于其蛋白酶抑制活性来抑制血管生成，而肝素结合部位对于发挥其抗血管生成活性是重要的

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