Study for clinical use of anti-inetastatic agent, bikunin

我们先前在一系列文献中报道了一种库尼茨类型的蛋白酶抑制剂bikunin，它可以抑制尿激酶型纤溶酶原激活物(UPA)及其特异性受体(UPAR)的表达上调，抑制ERK1/2的磷酸化，从而抑制体外癌细胞的侵袭和体内的腹膜播散性转移。本研究探讨了大豆胰蛋白酶抑制剂(SBTI)对人卵巢癌细胞株HRA分泌的胞外uPA的净酶活性、uPA表达的信号转导及侵袭力的影响。SBTI含有一种库尼茨胰酶抑制剂(KTI)和一种Bowman-Birk抑制剂(BBI)。在这里，我们发现：1)KTI和BBI是从大豆中分离纯化的，2)KTI和BBI都不能有效地抑制uPA的酶活性，3)KTI与~2μM的IC_(50)&gt；预先与细胞孵育可抑制hRA细胞uPA的上调，而BBI未能抑制uPA的上调，通过酶联免疫吸附试验测定，4)细胞的侵袭力被KTI与IC_(50)&GT；体外侵袭实验表明，BBI不能抑制μ细胞的侵袭；5)KTI通过阻断uPA的上调而抑制细胞的侵袭，这种上调可能是由比库宁结合蛋白和/或其受体以外的结合蛋白(S)介导的；6)转化生长因子-β1(β-1)介导的ERK1/2的活化被KTI预先孵育后显著降低。总之，KTI，而不是BBI，至少可以通过抑制uPA信号级联来抑制细胞侵袭，尽管KTI的机制可能与bikunin不同。

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.