Study for clinical use of anti-inetastatic agent, bikunin

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We have previously reported in a series of papers that a Kunitz-type protease inhibitor, bikunin, suppresses up-regulation of urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) expression, phosphorylation of ERK1/2 and cancer cell invasion in vitro and peritoneal disseminated metastasis in vivo. In the present study, we investigated the effects of soy bean trypsin inhibitor (SBTI) on the net enzymatic activity of secreted, extracellular uPA, signal transduction involved in the expression of uPA and invasion in HRA human ovarian cancer cells. SBTI contains a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk inhibitor (BBI). Here, we show 1) that KTI and BBI were purified separately from soybeans, 2) that neither KTI nor BBI effectively inhibits enzymatic activity of uPA, 3) that uPA upregulation observed in HRA cells was inhibited by preincubation of the cells with KTI with an IC_<50> of 〜2μM, whereas BBI failed to repress uPA upregulation, as measured by enzyme-linked immunosorbent assay, 4) that cell invasiveness was inhibited by treatment of the cells with KTI with an IC_<50> of 〜3μM, whereas BBI failed to suppress cell invasion, as measured by an in vitro invasion assay, 5) KTI suppresses HRA cell invasion by blocking uPA up-regulation which may be mediated by a binding protein(s) other than a bikunin binding protein and/or its receptor, and 6) that transforming growth factor-beta 1 (TGF-β1)-mediated activation of ERK1/2 was significantly reduced by preincubation of the cells with KTI. In conclusion, KTI, but not BBI, could inhibit cell invasiveness at least through suppression of uPA signaling cascade, although the mechanisms of KTI may be different from those of bikunin.