Effect of protein C inhibitor on renal cell carcinogenesis and on tumor invasion, growth and metastasis

蛋白C抑制物(PCI)是丝氨酸蛋白酶抑制物(Serpin)家族的一员，它调节抗凝蛋白C途径，抑制肿瘤细胞侵袭的介质--尿纤溶酶原激活物(UPA)。我们最近证实，在来源于肾近端小管上皮细胞的肾癌细胞中，pCI的表达显著降低，并且pCI通过抑制uPA而在体外抑制肾癌细胞的Matrigel侵袭。此外，我们还认为，在肾细胞癌中，pCI的表达降低主要是由于pCI基因启动子区甲基化所致。在本研究中，我们阐明了pCI基因甲基化导致肾癌中pCI表达下调的详细机制，并利用人乳腺癌细胞(MDA-231)评价了人pCI及其非活性衍生物对肿瘤细胞体外侵袭和体内生长转移的影响。亚硫酸氢盐的dna序列显示其中4个…在肾细胞癌的pCI基因中，位于转录起始点附近的13个CpG序列发生了特异性甲基化。利用甲基化的pCI基因片段对荧光素酶报告基因的分析表明，pCI基因启动子片段的转录活性因这四个CpG序列在转录起始点附近的甲基化而降低。另一方面，表达完整pCI的MDA-231细胞(mda-pCI)的体外侵袭力显著低于表达非活性R354ApCI的mda-231细胞(mda-R354apci)或表达pCI的N-末端片段的mda-231细胞(mda-NTpCI)。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。综上所述，这些研究表明，在肾癌中，pCI表达降低主要是由于pCI基因启动子的特异性CpG甲基化引起的，除了其反应性的部位依赖作用外，pCI还调节肿瘤的生长和转移，而不依赖于它的蛋白水解酶抑制活性。较少

Protein C inhibitor (PCI), a member of the serine protease inhibitor (serpin) family, regulates the anticoagulant protein C pathway and inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. We recently demonstrated that PCI expression is significantly decreased in renal carcinoma cells derived from renal proximal tubular epithelial cells, and that PCI suppresses in vitro Matrigel invasion of renal carcinoma cells by inhibiting uPA. Furthermore, we also suggested that decreased expression of PCI in renal cell carcinoma (RCC) is mainly caused by methylation of the promoter region of PCI gene. In the present study, we elucidated the detailed mechanism of decreased expression of PCI induced by PCI gene methylation in RCC, and evaluated the effect of human PCI and its inactive derivatives on tumor cell invasion in vitro, and on tumor growth and metastasis in vivo using human breast cancer (MDA-231) cells. Hydrogen bisulfite DNA sequence showed that four out of thi … More rteen CpG sequence located near transcription initiation site are specifically methylated in PCI gene of RCC. Luciferase reporter gene assay using methylated PCI gene fragment showed that transcriptional activity of PCI gene promoter fragment is decreased by methylation of these four CpG sequence near transcription initiation site. On the other hand, the in vitro invasiveness of MDA-231 cells expressing intact PCI (MDA-PCI) was significantly decreased as compared to MDA-231 cells expressing inactive R354APCI (MDA-R354APCI) or the N-terminal fragment of PCI (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. Overall, these studies showed that, decreased PCI expression in RCC is mainly caused by specific CpG methlation of PCI gene promoter, and in addition to its reactive site-dependent action, PCI also regulates tumor growth and metastasis independently of its protease inhibitory activity. Less