The surface of prostate carcinoma DU145 cells mediates the inhibition of urokinase-type plasminogen activator by maspin

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor inva sion and metastasis, It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility, Interestingl y, 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 speci fically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-Lysine, the molecular mechanism underlying the tumor-s uppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our exp erimental system, we chose prostate carcinoma DU145 cells because these cel ls mediate plasminogen activation primarily by uPA, as shown by two differe nt colorimetric enzyme activity assays. Purified recombinant maspin produce d in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i )] binds specifically to the surface of DU145 cells, inhibits the DU145 cel l surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate, The inhibitory effect of rMaspin(i) on fell surface-bound uPA was s imilar to that of an uPA-neutralizing antibody and was reversed by a polycl onal antibody against the reactive site loop sequence of maspin. The K-i va lue for rMaspin(i) in cell surface-mediated plasminogen activation was 20 n M, which was comparable to the K-i values for plasminogen activator inhibit or 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent w ith its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in m ediating the inhibitory interaction between rMaspin(i) and uPA, Thus, futur e maspin-based therapeutic strategies may prove useful in blocking the inva sion and metastasis of uPA-positive prostate carcinoma.