Treatment with beta-glucan may be beneficial for cancer patients with or at risk for metastasis. The beta-glucan-dependent signaling pathways are critical for our understanding of anticancer events and development of cancer therapeutic agents.

The modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean trypsin inhibitor, as dietary supplements on experimental and spontaneous pulmonary metastasis of murine Lewis lung carcinoma 3LL cells as well as peritoneal disseminated metastasis model in human ovarian cancer HRA cells were investigated in i.v., s.c. and i.p. injection models in mice. Seven groups of female C57BL/6 or nude mice were fed a basal diet (control group) or the basal diet supplemented with KTI or BBI (5, 15, or 50 g/kg). Here we show that, in an in vivo spontaneous metastasis assay, the diet supplementation with KTI (15 and 50 g/kg), but not with BBI, for 28 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of KTI. In an in vivo experimental metastasis assay, the diet supplementation with KTI or BBI for 21 days after i. Key words: dietary supplementation; soybean; trypsin inhibitor; metastasis; urokinaseThe invasion and metastasis of tumor cells from a primary lesion to distant sites in a target organ are multistep dynamic processes. Of these, the invasiveness of tumor cells into extracellular matrices (ECMs) represents one of several important properties necessary for the formation of metastasis. 1 The tumor cells secrete various specific proteolytic enzymes to invade through ECMs. Various test systems devised to investigate the mechanisms of tumor invasion and inhibition of experimental metastasis in vitro have suggested that protease inhibitors for urokinase-type plasminogen activator (uPA), plasmin and matrix metalloproteases (MMPs) prevent tumor cell invasion to chick chorioallantoic membrane, 2,3 human amnion 4 and reconstituted basement membranes. 5 Evidence for the role of an uPA-plasmin-MMP activation cascade in cancer cells has been provided. 6,7 We have previously analyzed the effects of human amniotic fluid-or human urine-derived Kunitz-type protease inhibitor, bikunin, in detail and confirmed that bikunin is useful for the treatment of mouse and human cancer metastasis. 8 However, it becomes difficult to collect materials for purification of bikunin. Proteins of soybeans are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin. The 2 major classes of inhibitors are the Kunitz trypsin inhibitor (KTI), which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. 9,10 One gram of soybeans contains about 50 units of trypsin inhibitory activities (data not shown). Therefore, we recently examined the effects of soybean trypsin inhibitor (SBTI) on the net enzymatic activity of secreted extracellular uPA in human ovarian cancer HRA cells to assess the potential use of protease inhibitors in human cancer prevention and ...

We have previously reported in a series of reports that a Kunitz-type protease inhibitor, bikunin derived from human urine (also known as urinary trypsin inhibitor), suppresses expression of uPA, phosphorylation of ERK1/2, and cancer cell invasion in vitro and peritoneal disseminated metastasis and lung metastasis in vivo (1-5). More recently, we investigated the effects of soybean trypsin inhibitor on the uPA expression, signal transduction involved in the expression of uPA, and invasion in human ovarian cancer HRA cells (6, 7). Soybean trypsin inhibitor contains a Kunitz trypsin inhibitor (KTI) 1 and a Bowman-Birk inhibitor (BBI) (8). We previously showed (6, 7) that 1) uPA expression observed in HRA cells is inhibited by preincubation of the cells with KTI with an IC 50 of ϳ2 M, whereas BBI failed to repress uPA expression; 2) cell invasiveness is inhibited by treatment of the cells with KTI with an IC 50 of ϳ3 M, whereas BBI failed to suppress cell invasion; 3) KTI suppresses HRA cell invasion by blocking uPA up-regulation, which may be mediated by one or more binding proteins other than a bikunin-binding protein and/or its receptor; and 4) TGF-␤1-mediated activation of ERK1/2 is significantly reduced by preincubation of the cells with KTI. We conclude that KTI, but not BBI, could inhibit cell invasiveness at least through suppression of the MAPK-dependent uPA signaling cascade (6, 7).It has been reported that there appeared to be a specific interaction between bikunin and the tumor cell surface (9 -12). Tumor cells express two types of bikunin-binding proteins; a 40-kDa bikunin-binding protein, which is identical to cartilage link protein, and a 45-kDa bikunin-binding protein, a putative bikunin receptor (12, 13). Bikunin binds link protein and bikunin receptor on tumor cell surface possibly via the Nterminal Kunitz domain I and the chondroitin sulfate side chain, respectively (13). Bikunin must bind directly to both of the cell-associated bikunin-binding proteins to suppress expression of uPA and uPA receptor genes (14). However, KTI

Our results indicate that KTI inhibits LPS-induced up-regulation of cytokine expression possibly through suppression of ERK1/2 and p38 kinase-mediated NFkappaB activation. These findings may identify anti-inflammatory properties of KTI at the level of gingival fibroblasts and may be relevant to the use of KTI in modulating inflammation, including periodontal disease.

We examined the modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean, as intraperitoneal (i.p.) injection and dietary supplements on bacterial lipopolysaccharide (LPS)-induced lethality in mice. We initially examined the suppressing effects of i.p. injection of KTI (50 mg/kg) and BBI (50 mg/kg) on LPS-induced lethality after i.p. injection of LPS. Furthermore, groups of female C57BL/6 were fed a basal diet (control group) or the basal diet supplemented with KTI (50 g/kg) or BBI (50 g/kg). Here, we show that i.p. and daily oral administration of KTI, but not BBI, caused a significant reduction of the LPS-induced lethality; that LPS significantly induced plasma TNF-alpha, IL-1beta, and IL-6 levels in mice after LPS challenge; that concomitant administration of KTI, but not BBI, inhibits the LPS-induced plasma levels of these cytokines; and that KTI, but not BBI, suppressed LPS-induced upregulation of cytokine expression through suppression of phosphorylation of three mitogen-activated protein (MAP) kinase pathways, ERK1/2, JNK, and p38, in peritoneal macrophages. These data allow us to speculate that i.p. injection and dietary supplementation of a soybean KTI may play a role as a potent anti-inflammatory agent by inhibiting activation of MAP kinases, leading to the suppression of cytokine expression.