Study on the mechanism of extracellular matrix degradation induced by tumor cell associated fibrinolytic enzyme

基底膜和细胞外基质的溶解和迁移是肿瘤细胞的关键步骤。纤溶酶系统如尿激酶型纤溶酶原激活物(UPA)及其抑制物(PAI)在肿瘤的侵袭和转移中起重要作用。UPA能与肿瘤细胞膜表面的uPA受体结合，与PAI形成复合体(uPA/PAI复合体)。UPA/PAI复合体不具有纤溶活性，但体外仅有PA抑制活性。但肿瘤细胞中高水平的uPA提示肿瘤患者预后不良。这一事实似乎是矛盾的。我们通过流式细胞仪和纤维蛋白平板实验证明，uPA/PAI-1复合体与大量活化蛋白C(APC)孵育后，细胞膜上的uPA活性重新出现。将50 nM的uPA和50 nM的PAI-1在室温(RT)孵育30min后形成uPA/PAI-1复合体，然后在RT下加入30倍摩尔的APC孵育30min。UPA/PAI-1复合体没有纤溶活性，但与APC孵育后显示出类似uPA的纤溶活性。本研究还证明了APC与UPA/PAI-1复合体孵育后可形成APC/PAI-1复合体，并抑制PAI-1的活性。这些事实表明，即使在与PAI-1形成复合体后，肿瘤细胞相关的uPA活性也是可逆的，并可以降解细胞外基质。

Dissolution of and migration through basement membranes and extracellular matrices are critical steps for tumor cells. The fibrinolytic enzyme system such as urokinase type plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor, PAI) seems to play an important role in tumor invasion and metastasis formation. The uPA,which can bind uPA receptor on the surface of tumor cell membrane, can make a complex with PAI (uPA/PAI complex). The uPA/PAI complex has no fibrinolytic activity, but has only PA inhibiting activity in vitro study. But high amounts of uPA in tumor cells show poor prognosis in patients with carcinoma. This fact seems contradictory.We proved that uPA activity of cell membrane reappear after incubation with uPA/PAI-1 complex a large amount of activated Protein C (APC) by flow cytometry and in fibrin plate assay. The uPA/PAI-1 complex was formed after 30 min incubation of 50 nM uPA and 50 nM PAI-1 at room temperature (RT), and then 30-fold molar of APC was added to the complex at RT for 30 min. The uPA/PAI-1 complex had no fibrinolytic activity, but after the incubation with APC the complex showed the fibrinolytic activity like uPA.In this study, we also proved APC could make APC/PAI-1 complex after incubation of uPA/PAI-1 complex with APC,and the activity of PAI-1 was inhibited. These facts show tumor cell associated uPA activity can be reversible even after the complex formation with PAI-1, and can degrade extracellular matrix.