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Background: Human bikunin, a Kunitz-type trypsin inhibitor, inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in tumor cells and inflammatory cells., Objectives: We analyzed the effect of a soybean-derived Kunitz trypsin inhibitor (KTI) on TNF-alpha production in human gingival fibroblasts stimulated by lipopolysaccharide (LPS), an inflammatory inducer., Material and Methods: Mitogen-activated protein kinase (MAPK) activation and cytokine levels were monitored using western blot and a specific enzyme-linked immunosorbent assay (ELISA)., Results: Here, we show (i) a soybean KTI abrogates LPS-induced up-regulation of TNF-alpha mRNA and protein expression in a dose-dependent manner in gingival fibroblasts, (ii) KTI also blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins, (iii) inhibition by KTI of TNF-alpha induction correlates with the suppressive capacity of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 signaling pathways, implicating repressed ERK1/2 and p38 signalings in the inhibition, and (iv) pretreatment of cells with KTI blocked LPS-induced nuclear factor kappaB (NFkappaB) activation., Conclusion: 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.

Background: Cytokines are produced as a consequence of photo-damaged DNA and oxidative stress in ultraviolet (UV)-exposed keratinocytes. A soybean Kunitz trypsin inhibitor (KTI) down-regulates the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in tumor cells and inflammatory cells., Aim: The effect of KTI on TNF-alpha production in UV-exposed primary human keratinocytes was analyzed., Results: We show (i) UV induced up-regulation of TNF-alpha mRNA and protein expression in keratinocytes; (ii) cells treated with KTI before UV irradiation showed a significantly lower accumulation of TNF-alpha protein in a dose-dependent manner and a reduced UV-induced up-regulation of TNF-alpha mRNA expression; (iii) KTI inhibited the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins; (iv) UV irradiation transiently activated c-Jun N-terminal kinase (JNK) and Akt signaling but only weakly activated extracellular signal-regulated kinase (ERK) and p38; (v) KTI specifically inhibited UV-induced activation of ERK, JNK, and p38, but not Akt; (vi) treatment of cells with SP600125, a pharmacological inhibitor of JNK, predominantly suppressed UV-induced up-regulation of TNF-alpha expression; and (vii) KTI did not enhance suppression of UV-induced JNK phosphorylation by SP600125., Conclusions: KTI specifically inhibited UV-induced up-regulation of cytokine expression predominantly through suppression of JNK signaling pathway.

A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-beta1 (TGF-beta1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-beta1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-beta1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-beta1-induced phosphorylation of Src, ERK1/2, and Akt by 40-60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-beta1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-beta1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src.

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.

We provided evidence previously that bikunin, a Kunitz-type protease inhibitor, can disrupt dimerization of CD44 proteins, which may result in suppression of receptor-mediated MAP kinase signaling. However, to what extent dimerization may alter ligand-induced signaling has not been documented. Given the recent recognition that some growth factor receptors can form heterodimers with CD44, the present study was undertaken to determine whether the CD44 and growth factor receptors (e.g., EGFR, FGFR, HGFR, VEGFR, TGF-betaRI, or TGF-betaRII) can form heterodimers in cancer cells and, if so, to investigate the potential functional consequences of such heterodimerization. We also examined whether bikunin can abrogate these heterodimerizations and inhibit CD44/growth factor-dependent signaling. Here, we show direct evidence for heterodimerization of CD44-FGFR and CD44-TGF-betaRI in human chondrosarcoma HCS-2/8 cells, CD44-EGFR complex in human glioma U87MG cells, and CD44-TGF-betaRI heterodimer in human ovarian cancer HRA cells. Coupling of CD44 and growth factor receptor may be selective, depending on a cell type. Bikunin does not alter the ligand binding, whereas functionally reduces heterodimerization between CD44 and growth factor receptors. The disruption of heterodimerization substantially reduces receptor-induced tyrosine phosphorylation and ERK1/2 activation. Taken together, our data suggest that bikunin-mediated suppression of heterodimerization between CD44 and growth factors may inhibit the agonist-promoted activation of the signaling pathway., ((c) 2005 Wiley-Liss, Inc.)

Purpose: Bikunin is a multifunctional glycoprotein, which mediates suppression of tumor cell invasion and metastasis. The measurement of bikunin levels in the tissue of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the evaluation of prognosis. The high bikunin expression in ovarian cancer tissue would enable the use of soluble bikunin protein present in the circulation of ovarian cancer patients as a biomarker of disease., Patients and Methods: We developed a double-antibody immunoassay for bikunin and detected its presence in normal human circulation. We quantified, by enzyme-linked immunosorbent assay and/or immunoblot assay bikunin in sera from 200 healthy women (controls), 200 patients with benign gynecologic diseases, and 327 patients with ovarian cancer before surgical removal of the tumor., Results: When the values of bikunin corresponding to the median were used as the cutoff value (11.5 microg/mL), low plasma bikunin was strongly associated with late-stage, suboptimal debulking with large residual tumor (> 2 cm) and low response to chemotherapy. The median survival time of the patients with a high bikunin level was more than 60 months as compared with 26 months among those with low bikunin level (P = .002). This difference corresponded to a 2.2-fold increased risk of dying for the lower plasma bikunin patients (hazard ratio, 0.45; P = .023) and remained significant in multivariate analysis (hazard ratio, 0.63; P = .041)., Conclusion: Preoperative plasma bikunin concentration is a strong and independent favorable prognostic marker for ovarian cancer.

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.v. tumor cell inoculation did not reduce the number of lung tumor colonies. In addition, KTI (15 or 50 g/kg) treatment in a peritoneal disseminated metastasis model of HRA cells resulted in a 40% reduction in total tumor burden when compared with control animals. Immunoblot analysis revealed that KTI specifically reduced expression of uPA protein as well as phosphorylation of MAP kinase and PI3 kinase proteins in the cells stimulated with agonists (G-CSF for 3LL cells or TGF-beta1 for HRA cells). These results suggest that dietary supplementation of KTI more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation during the metastatic process. KTI treatment may also be beneficial for ovarian cancer patients with or at risk for peritoneal disseminated metastasis; it greatly reduces tumor burden in part by inhibiting phosphorylation of MAP kinase and PI3 kinase, leading to suppression of uPA expression.

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-beta1 (TGF-beta1)-stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-beta1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-beta1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-beta receptors (TbetaRI and TbetaRII) in either cell types nor altered the specific binding of 125I TGF-beta1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TbetaRI and TbetaRII present on the same cells exclusively form aggregates in TGF-beta1-stimulated cells; (e) co-treatment of TGF-beta1-stimulated cells with bik suppresses TGF-beta1-induced complex formation of TbetaRI and TbetaRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-beta1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-beta1-elicited signaling cascades in cells possibly by abrogating the coupling between TbetaRI and TbetaRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik(-/-) female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

Objective: This study was carried out to clarify the localization of bikunin, a Kunitz-type protease inhibitor, and relation between expression of individual bikunin protein and ovarian cancer progression., Methods: We performed a retrospective study on the immunohistochemical expression of bikunin, urokinase-type plasminogen activator (uPA) and macrophages (CD68) in surgical specimens derived from 89 ovarian cancer patients to investigate correlations between the expression of bikunin and the clinicopathologic features and the prognosis. Furthermore, bikunin and uPA levels were measured by immunoblot analysis., Results: Immunohistochemical staining revealed that the localization of bikunin was similar to that of CD68 for macrophages. We identified high expression of bikunin in 40 (45%) of 89 ovarian cancers. The results of Western blot analysis showed a significant correlation with immunohistochemical data. There was a significant inverse correlation between bikunin levels and uPA levels in ovarian cancer tissues. High bikunin expression was an independent predictor for disease-free survival (P = 0.040) and overall survival (P = 0.042). The 5-year survival rate of the 49 patients with low bikunin expression in ovarian cancers was 39%, whereas that of the other 40 patients with high bikunin expression was 63%. In addition, macrophage-derived bikunin protein was induced by exogenous IL-6., Conclusion: Bikunin derived from tumor-infiltrating macrophages might be a prognostic indicator as an antiinvasive factor supplied from macrophages within and around the tumor possibly through down-regulation of tumor-associated uPA expression.

Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of (125)I-bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once-daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose-dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis-related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once-daily i.p. administration of bikunin (25 microg/g body weight/day) in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression., (Copyright 2004 Wiley-Liss, Inc.)

A Kunitz-type protease inhibitor, bikunin, downregulates expression of uPA and its receptor uPAR at the mRNA and protein levels in several types of tumor cells. Our recent work showed that, using a cDNA microarray analysis, pregnancy-associated plasma protein-A (PAPP-A) is a candidate bikunin target gene. To clarify how reduced levels of PAPP-A may confer repressed invasiveness, we transfected human ovarian cancer cell line HRA with antisense (AS)-PAPP-A cDNA and compared the properties of the transfected cells to those of parental HRA cells. Here, we show that regulation of uPA mRNA and protein by IGF-I depends on the PI3K and MAPK signaling pathways and phosphorylation of Akt and ERK1/2 is required for IGF-I-mediated cell invasion; that IGFBP-4 protease in HRA cells is identified as PAPP-A; that reduced PAPP-A expression is associated with the upregulation of IGFBP-4 expression; that higher intact IGFBP-4 levels were associated with low invasive potential and growth rate in AS-PAPP-A cells in response to IGF-I; that IGF-I stimulates Akt and ERK1/2 activation of both the control and antisense cells, but the relative potency and efficacy of IGF-I were lower in the antisense cells compared to the control; and that genetic downregulation of PAPP-A reduces the proliferation, invasion and metastasis of HRA cells. In conclusion, our data identify a novel role for PAPP-A as a bikunin target gene. IGF-I-induced IGFBP-4 proteolysis by PAPP-A may enhance cell growth and invasion through IGF-I-dependent Akt and ERK1/2 activation and subsequently upregulation of uPA., (Copyright 2004 Wiley-Liss, Inc.)

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.

Background: Bikunin, a Kunitz-type protease inhibitor, specifically inhibits tumor invasion and metastasis., Methods: The authors initially evaluated the therapeutic efficacy of once-daily oral administration of different doses of bikunin against human ovarian carcinoma HRA cells growing in the peritonea of nude mice. For the in vivo studies, female 7-week-old nude mice were randomized to 1 of 4 groups: bikunin-treated groups (n = 9 in each group) received 3, 10, or 30 microg/g body weight per day bikunin for 7 days via gastrointestinal gavage, and a control group (n = 9) received the vehicle solution (phosphate-buffered saline) via gastrointestinal gavage. On Day 9, the abdominal cavity was examined by two observers who were blinded to treatment., Results: After oral administration, intact bikunin was detectable in mouse serum specimens at 3 and 6 hours. This was followed by a decline at 12 hours. The mice given bikunin at the highest dose level had a 40% decrease in tumor load. The highest uptake in the tumor was obtained with [125I]bikunin 12 hours postadministration. No effect on either food intake or body weight was observed in the treated versus sham groups. The current study was the first to report the potent activity of once-daily oral administration of bikunin against ovarian carcinoma. Next, the authors performed a Phase I trial to determine the maximum-tolerated dose (MTD) and safety of a once-daily oral administration schedule. The indication was locally advanced uterine cervical carcinoma after definitive treatment. An escalating dose (3, 10, and 30 mg/kg per day) of bikunin was administered orally to nine patients for 7 days. There were no dose-limiting toxicities and the MTD of the bikunin schedule was not defined. The authors also obtained preliminary data on its effect on urokinase-type plasminogen activator expression at the highest dose level., Conclusions: Once-daily oral administration of bikunin was found to be safe in humans and exhibited signs of biologic activity., (Copyright 2004 American Cancer Society.)

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 approximately 2 microM, 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 approximately 3 microM, 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-beta1)-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.

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.

Background: In previous studies, the authors showed that various types of cultured tumor cells treated with exogenous bikunin protein or ovarian carcinoma cells transfected with bikunin cDNA have low invasiveness and diminished metastatic potential. This study was carried out to clarify the relation between the expression of individual bikunin mRNA and tumor progression., Methods: Forty-one newly diagnosed ovarian carcinomas were investigated using a semiquantitative reverse transcriptase-polymerase chain reaction., Results: The authors found that 24 patients had tumors that overexpressed bikunin and that gene expression was reduced in the tumors of the remaining 17 individuals. Bikunin mRNA expression was independent of age, surgical stage, tumor size, degree of differentiation, histologic subtype, and serum CA 125 levels. There was a significant correlation between low expression of bikunin mRNA and lymph node status (P=0.035) or peritoneal status (P=0.042). Multivariate analysis indicated that bikunin was an independent prognostic marker (P=0.013; hazard ratio, 2.30; 95 % confidence interval, 1.13-4.19), even after controlling for lymph node metastasis and the degree of peritoneal dissemination. In addition, low expression was a significant predictor for poor prognosis compared with high expression (2-year survival rate; 75.0 % vs. 47.1 %, respectively; P<0.05)., Conclusions: The data suggest that low bikunin mRNA expression by ovarian carcinoma cells may be associated with poor prognosis. It is conceivable that testing for bikunin mRNA may identify patients with ovarian carcinoma who are at high risk for early disease recurrence and a poor prognosis., (Copyright 2003 American Cancer Society.)

Bikunin, a Kunitz-type protease inhibitor, could potentially suppress tumor cell invasion and metastasis. Our previous study revealed that overexpression of bikunin in a human ovarian cancer cell line, HRA, resulted in a down-regulation in uPA and uPAR gene expression. For identifying the full repertoire of bikunin-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from bikunin-treated or bikunin-transfected HRA cells. A number of bikunin-regulated genes were identified, and their regulation was confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified genes can indeed be classified into distinct subsets. These include transcriptional regulators, oncogenes/tumor suppressor genes, signaling molecules, growth/cell cycle, invasion/metastasis, cytokines, apoptosis, ion channels, extracellular matrix proteins, as well as some proteases. This screen identified suppression of several genes such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronan-binding protein, matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which have previously been implicated in enhancing tumor promotion. Northern blot analysis confirmed that several genes including matriptase and PAPP-A were down-regulated by bikunin by approximately 9-fold. Further, genetic inhibition of matriptase or PAPP-A could lead to diminished invasion. These results show that bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion.

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik(+) clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik(+) clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik(+) clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik(+) clones., (Copyright 2003 Wiley-Liss, Inc.)

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.

To investigate the specific components involved in regulating cumulus cell-oocyte complex (COC) expansion in an in vitro mouse experiment, freshly-isolated COC were cultured in the presence of various combinations of FSH (1.0 microg/ml), proteins of the inter-alpha-inhibitor (I alpha I) family (a light chain, also known as bikunin, heavy chains [HC1 + HC2] and I alpha I [0.01-2.0 microg/ml]) and link protein (LP) (0.016-10 microg/ml) for 24 h and were observed for expansion of their cumulus cells (percent of COC with + 3 and + 4 expansion and average projected area). The COC were videotaped in real time at the initiation of culture and after 24 h of culture. FSH alone did not stimulate cumulus expansion under serum-free conditions; however, treatment with I alpha I (0.1-2.0 microg/ml) or heavy chains (10 microg/ml), but not bikunin (10 micro g/ml), in the presence of FSH significantly increased COC expansion, with maximal promotion occurring at 1.0 microg/ ml of I alpha I. Addition of LP (2.0 micro g/ml) to the medium containing I alpha I (1.0 microg/ml) and FSH resulted in significantly higher expansion levels than were observed in response to I alpha I alone, although LP alone (10 microg/ml) had no or very little effect by itself. Anti-I alpha I or anti-LP polyclonal antibody, which inhibits binding of I alpha I and LP, respectively, to hyaluronic acid (HA), markedly reduced expansion of the surrounding cumulus cell extracellular matrices. Therefore, in vitro, LP might serve, in part, to enhance the COC expansion possibly by stabilizing HA-I alpha I (or heavy chains) complex on the surrounding cumulus cell matrices., (Copyright 2002 Wiley-Liss, Inc.)

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA; (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, approximately 150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.

The protease inhibitor part of inter-alpha trypsin inhibitor is identical to urinary trypsin inhibitor (UTI). Preincubation of neutrophils and HUVEC cells with UTI inhibited increase of cytosolic free Ca2+ induced by LPS. Increase of cytosolic free Ca2+ induced by LPS in the presence of EGTA was also inhibited by UTI. In contrast, UTI did not inhibit increase of cytosolic free Ca2+ in cells stimulated by Ca2+ ionophore with or without EGTA. The effects of nine synthetic peptides of UTI on the concentration of cytosolic free Ca2+ in the neutrophils induced by LPS were examined. Preincubation with a peptide of UTI domain 2, NLPIVRGPCRAFIQL (83-97), was completely inhibited by the increase of cytosolic free Ca2+ in neutrophils. This region is identical to the trypsin inhibitor site of UTI. We propose that a function of UTI other than as a protease inhibitor is in regulation of intracellular Ca2+ and that this is due to its trypsin inhibitor region.

Urinary trypsin inhibitor (UTI) is one of the Kunitz-type protease inhibitors in human. Little is known about its anti-inflammatory functions other than protease inhibition. We studied the effect of UTI on gene expression of interleukin-8 (IL-8), an inflammatory cytokine. UTI inhibited IL-8 gene expression induced by lipopolysaccharide (LPS) in HL60 cells. The IL-8 concentrations in the cells and medium after LPS stimulation increased time-dependently in the absence of UTI, but did not increase in the presence of UTI. On the other hand, UTI did not inhibit either the synthesis or the release of IL-8 induced by the calcium ionophore A23187. UTI inhibited increase of cytosolic Ca2+ stimulated by LPS but not by A23187. Our results suggest that the inhibition by UTI is due to its effect on the cell membrane involved in regulating Ca2+ influx.