McGowen R, Biliran H, Sager R, Sheng S
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Cancer Res. 2000 Sep 1;60(17):4771-8.
Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility. Interestingly, in cell-free solutions, maspin does not inhibit several serine proteases including tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). Despite the recent biochemical evidence that maspin specifically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-lysine, the molecular mechanism underlying the tumor-suppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental system, we chose prostate carcinoma DU145 cells because these cells mediate plasminogen activation primarily by uPA, as shown by two different colorimetric enzyme activity assays. Purified recombinant maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reactive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was comparable to the Ki values for plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinoma.
Maspin是一种新型丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），在乳腺癌和前列腺癌中具有肿瘤抑制潜力，作用于肿瘤侵袭和转移水平。随后证明，maspin至少部分通过抑制细胞运动来抑制肿瘤侵袭。有趣的是，在无细胞溶液中，maspin不抑制包括组织型纤溶酶原激活剂和尿激酶型纤溶酶原激活剂（uPA）在内的几种丝氨酸蛋白酶。尽管最近有生化证据表明maspin特异性抑制与纤维蛋白原或聚-L-赖氨酸相关的组织型纤溶酶原激活剂，但maspin肿瘤抑制作用的分子机制仍然难以捉摸。本研究的目的是研究maspin对细胞表面相关uPA的影响。在我们的实验系统中，我们选择了前列腺癌DU145细胞，因为如两种不同的比色酶活性测定所示，这些细胞主要通过uPA介导纤溶酶原激活。在杆状病毒感染的草地贪夜蛾Sf9昆虫细胞中产生的纯化重组maspin [rMaspin(i)]特异性结合DU145细胞表面，抑制DU145细胞表面结合的uPA，并与DU145细胞裂解物中的uPA形成稳定复合物。rMaspin(i)对细胞表面结合的uPA的抑制作用与uPA中和抗体相似，并被针对maspin反应位点环序列的多克隆抗体逆转。rMaspin(i)在细胞表面介导的纤溶酶原激活中的Ki值为20 nM，分别与纤溶酶原激活剂抑制剂1和纤溶酶原激活剂抑制剂2的Ki值相当。此外，rMaspin(i)的蛋白水解抑制作用在数量上与其对体外DU145细胞运动的抑制作用一致。我们的数据证明了前列腺癌细胞表面在介导rMaspin(i)与uPA之间的抑制性相互作用中的重要作用。因此，未来基于maspin的治疗策略可能被证明对阻断uPA阳性前列腺癌的侵袭和转移有用。
