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A soybean Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase upregulation


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 IC50 of ∼ 2 μM, 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 IC50 of ∼ 3 μM, 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.

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  1. 1.

    Reuning U, Magdolen V, Wilhelm O et al. Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis (review). Int J Oncol 1998; 13 (5): 893–906.

    PubMed  CAS  Google Scholar 

  2. 2.

    Kobayashi H, Shinohara H, Gotoh J et al. Anti-metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent. Br J Cancer. 1995; 72 (5): 1131–7.

    PubMed  CAS  Google Scholar 

  3. 3.

    Kobayashi H, Suzuki M, Tanaka Y et al. Suppression of urokinase expression and invasiveness by urinary trypsin inhibitor is mediated through inhibition of protein kinase C-and MEK/ERK/c-Jun-dependent signaling pathways. J Biol Chem 2001; 276 (3): 2015–22.

    PubMed  CAS  Article  Google Scholar 

  4. 4.

    Kobayashi H, Suzuki M, Kanayama N et al. Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade. Eur J Biochem 2002; 269 (16): 3945–57.

    PubMed  CAS  Article  Google Scholar 

  5. 5.

    Kobayashi H, Suzuki M, Hirashima Y et al. The protease inhibitor bikunin, a novel anti-metastatic agent. Biol Chem. 2003; 384 (5): 749–54.

    PubMed  CAS  Article  Google Scholar 

  6. 6.

    Brandon DL, Bates AH, Friedman M. ELISA analysis of soybean trypsin inhibitors in processed foods. Adv Exp Med Biol 1991; 289: 321–37.

    PubMed  CAS  Google Scholar 

  7. 7.

    Kennedy AR, Troll W, Kennedy AR (eds). Protease Inhibitors as Cancer Chemopreventive Agents, 65–91 1993, New York: Plenum Publishing Corp.

    Google Scholar 

  8. 8.

    Kennedy AR, Troll W, Kennedy AR (eds). Protease Inhibitors as Cancer Chemopreventive Agents, 9–64 1993, New York: Plenum Publishing Corp.

    Google Scholar 

  9. 9.

    Kennedy AR. Chemopreventive agents: protease inhibitors. Pharmacol Ther 1998; 78 (3): 167–209.

    PubMed  CAS  Article  Google Scholar 

  10. 10.

    Kobayashi H, Ohi H, Terao T. Prevention by urinastatin of cisdiamminedichloroplatinum-induced nephrotoxicity in rabbits: Comparison of urinary enzyme excretions and morphological alterations by electron microscopy. Asia Oceania J Obstet Gynaecol 1991; 17 (3): 277–88.

    PubMed  CAS  Google Scholar 

  11. 11.

    Birk Y. Protein proteinase inhibitors in legume seeds-overview. Arch Latinoam Nutr 1996; 44 (4 Suppl 1): 26S–30S.

    PubMed  CAS  Google Scholar 

  12. 12.

    Liener IE. Possible adverse effects of soybean anticarcinogens. J Nutr 1995; 125 (3 Suppl): 744S–750S.

    PubMed  CAS  Google Scholar 

  13. 13.

    Kobayashi H, Suzuki M, Tanaka Y et al. A Kunitz-type protease inhibitor, bikunin, inhibits ovarian cancer cell invasion by blocking the calcium-dependent transforming growth factor-beta 1 signaling cascade. J Biol Chem. 2003; 278 (10): 7790–9.

    PubMed  CAS  Article  Google Scholar 

  14. 14.

    Pusztai A, Watt WB, Steward JC. A comprehensive scheme for the isolation of trypsin inhibitors and the agglutinin from soybean seeds. J Agric Food Chem 1991; 39: 882–6.

    Article  Google Scholar 

  15. 15.

    Morrissey D, O'Connell J, Lynch D et al. Invasion by esophageal cancer cells: functional contribution of the urokinase plasminogen activation system, and inhibition by antisense oligonucleotides to urokinase or urokinase receptor. Clin ExpMetastasis 1999; 17: 77–85.

    CAS  Google Scholar 

  16. 16.

    Hirashima Y, Kobayashi H, Suzuki M et al. Transforming growth factor-beta1 produced by ovarian cancer cell line HRA stimulates attachment and invasion through an up-regulation of plasminogen activator inhibitor type-1 in human peritoneal mesothelial cells. J Biol Chem 2003; 278 (29): 26793–802.

    PubMed  CAS  Article  Google Scholar 

  17. 17.

    Albini A, Iwamoto Y, Kleinman HK et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987; 47 (12): 3239–45.

    PubMed  CAS  Google Scholar 

  18. 18.

    Shetty S, Idell S. Posttranscriptional regulation of urokinase receptor gene expression in human lung carcinoma and mesothelioma cells in vitro. Mol Cell Biochem 1999; 199 (1-2): 189–200.

    PubMed  CAS  Article  Google Scholar 

  19. 19.

    Bell SM, Connolly DC, Maihle NJ, Degen JL. Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases. Mol Cell Biol 1993; 13 (9): 5888–97.

    PubMed  CAS  Google Scholar 

  20. 20.

    Bell SM, Brackenbury RW, Leslie ND, Degen JL. Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters. J Biol Chem 1990; 265 (3): 1333–8.

    PubMed  CAS  Google Scholar 

  21. 21.

    Suzuki M, Kobayashi H, Tanaka Y et al. Structure and function analysis of urinary trypsin inhibitor (UTI): Identification of binding domains and signaling property of UTI by analysis of truncated proteins. Biochim Biophys Acta. 2001; 1547 (1): 26–36.

    PubMed  CAS  Google Scholar 

  22. 22.

    von Hofe E, Newberne PM, Kennedy AR. Inhibition of Nnitrosomethylbenzylamine-induced esophageal neoplasms by the Bowman-Birk protease inhibitor. Carcinogenesis 1991; 12 (11): 2147–50.

    PubMed  CAS  Google Scholar 

  23. 23.

    Lewin MR, Chowcat NL, Jayaraj AP, Boulos PB. Collagenase inhibition in colonic mucosa by proteinase inhibitors. Br J Exp Pathol 1986; 67 (4): 523–6.

    PubMed  CAS  Google Scholar 

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Correspondence to Hiroshi Kobayashi.

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Kobayashi, H., Suzuki, M., Kanayama, N. et al. A soybean Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase upregulation. Clin Exp Metastasis 21, 159–166 (2004).

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  • Kunitz
  • protease inhibitor
  • soybean
  • urokinase-type plasminogen inhibitor