Inhibitory Effect of a Conjugate between Human Urokinase and Urinary Trypsin Inhibitor on Tumor Cell Invasion in Vitro

Abstract

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.

The extracellular matrix protein ITIH5 is a novel prognostic marker in invasive node-negative breast cancer and its aberrant expression is caused by promoter hypermethylation

“…Furthermore, several lines of evidence suggest a potential role of ITIs in tumour biology, particularly in the metastatic cascade and tumour progression (Kobayashi et al, 1995;Bourguignon et al, 1999;Paris et al, 2002;Zhang et al, 2004). We have previously described cloning and RNA expression analysis of a novel member of the ITIH gene family, ITIH5, in normal and malignant breast tissues (Himmelfarb et al, 2004).…”

“…Interestingly, the results on the expression analysis of ITIH5 in normal and malignant breast tissue are in line with those of previous studies investigating in general the role of ITIs in epithelial tumours. A role of ITIs in tumour invasion has been postulated on the basis of their protease inhibitor function mediated by bikunin (Kobayashi et al, 1995), and their capacity to bind to the extracellular matrix component HA mediated by the heavy chains (Huang et al, 1993; Zhao et al, 1995). In a lung cancer mouse model, overexpression of ITI protein chains led to inhibition of tumour progression and metastatic spread (Bourguignon et al, 1999).…”

“…Various studies (Kobayashi et al, 1995;Bourguignon et al, 1999;Paris et al, 2002;Zhang et al, 2004) have shown involvement of ITIs in tumour biology using an expressed sequence-tag-based bioinformatics approach (Dahl et al, 2005). We have previously identified ITIH5 as a novel gene differentially expressed in breast cancer.…”

Adenovirus-mediated gene transfer of urokinase plasminogen inhibitor inhibits angiogenesis in experimental arthritis

“…The urokinase plasminogen activator (uPA) binds to its receptor uPAR by its light chain fragment, 14 known as amino-terminal fragment (ATF, residues 1-135). This interaction involves the EGF-like domain of ATF and is species-restricted.…”

A gene transfer comparative study of HSA-conjugated antiangiogenic factors in a transgenic mouse model of metastatic ocular cancer

“…[7][8][9][10] Similarly, a 20-kDa (184 amino acid (aa)) C-terminal fragment of collagen XVIII, termed endostatin (ES), is reported to be a potent inhibitor of tumor angiogenesis and micrometastases development. 5,11 The urokinase plasminogen activator (uPA) binds to its receptor uPAR via its light chain fragments, 12 known as amino-terminal fragment ATF (residues . This interaction involves the EGF-like domain of ATF and its derivatives.…”