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Proteolytic enzymes such as urokinase-type plasminogen activator (uPA), plasmin, and collagenase mediate proteolysis by a variety of tumor cells. uPA secreted by tumor cells can be bound to a cell surface receptor via a growth factor-like domain within the amino-terminal fragment (ATF) of the uPA molecule with high affinity. Urinary trypsin inhibitor (UTI) efficiently inhibits the soluble and the tumor cell-surface receptor-bound plasmin and subsequently reduces tumor cell invasion and the formation of metastasis. The anti-invasive effect is dependent on the anti-plasmin activity of the UTI molecule, domain II in particular. We synthesized a conjugate between ATF of human uPA and a native UTI molecule or domain II of UTI (HI-8). The effect of the conjugates (ATF.UTI or ATF.HI-8) on tumor cell invasion in vitro was investigated. ATF.UTI and ATF.HI-8 bound to U937 cells in a rapid, saturable, dose-dependent, and reversible manner. A large part of receptor-bound ATF-UTI and ATF.HI-8 remains on the cell surface for at least 5 h at 37 degrees C. Inhibition of tumor cell-surface receptor-bound plasmin by ATF.UTI and ATF.HI-8 was markedly enhanced when compared with tumor cells treated either with ATF, UTI, or HI-8. Results of a cell invasion assay showed that ATF.UTI and ATF.HI-8 is very effective at targeting HI-8 specifically to uPA receptor-expressing tumor cells, whereas tumor cells devoid of uPA receptor may be less affected by the conjugates. Our results indicate that cell surface uPA and plasmin activity is essential to the invasive process and that the conjugates exhibit plasmin inhibition to the close environment of the cell surface and subsequently inhibit the tumor cell invasion through Matrigel in an in vitro invasion assay.

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik ؉ clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik ؉ clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik ؉ clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik ؉ clones. © 2003 Wiley-Liss, Inc. Key words: bikunin; invasion; transfection; urinary trypsin inhibitor; urokinase-type plasminogen activatorInvasion and metastasis by tumor cells have multifactorial steps, may require the action of proteolytic enzymes at each step and are complex processes in which many gene activities are involved. 1 Various proteins on the surfaces of tumor cells mediate attachment to and degradation of the basement membrane and components of the extracellular matrix. 2 Increased activity, membrane association and secretion of urokinase-type plasminogen activator (uPA), plasmin, certain cathepsins and matrix metalloproteinases (MMPs) have been shown to correlate positively with invasiveness and the metastatic properties of many tumor entities. [1][2][3][4] The penetrating tumor cell focuses proteolytic activity at its cell surface through receptors for uPA, plasmin and MMPs. 3,4 uPA and its receptor are instrumental in cell invasion and metastasis. 5 uPA has a pleiotropic effect on cell migration and spreading in vivo and in vitro through the activation of plasminogen or other protein factors at the cell surface or in the extracellular matrix. Thus, uPA is at the center of a complex system affecting cell invasion and metastasis. 5 Bikunin (bik), also known as urinary trypsin inhibitor, is a Kunitz-type protease inhibitor which presents in human amniotic fluid, urine and in...