Purification of lectin and Kunitz trypsin inhibitor from soya seeds.

The search for potent and selective therapeutic agents is progressing by the study of natural compounds in plants. Plant-derived macromolecules are considered emerging therapeutic agents and an alternative to synthetic and small molecule drugs. Where it has long been known that plants possess medicinal properties, the compounds responsible for their action are in many cases still unknown: often only whole crude plant extracts or fractionated extracts are tested for the ability to inhibit common pathogens. Here, we present a fast protein liquid chromatography method for the separation of crude plant proteins. Kunitz trypsin inhibitor (KTI; 24.2 kDa) and lectin (31 kDa) were purified from Glycine max by liquid extraction followed by ion exchange column chromatography. The need for serial chromatographic separation steps has been eliminated by introducing more complex elution profiles hence reducing cost, time and improving recovery. The identity of KTI-A and lectin was confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS). Cell proliferation assays using B16F1 melanoma cells revealed that both KTI and the monomeric lectin retained some antiproliferative activity. This method could be useful for rapid and cost-effective purification of bioactive compounds from plant material.

BASI, a potent small molecular inhibitor, inhibits glioblastoma progression by targeting microRNA-mediated ß-catenin signaling.

BACKGROUND AND AIMS: The nuclear localization of ß-catenin, a mediator of canonical Wnt signaling, has been indicated in a variety of cancers and is frequently related to tumor progression and metastasis. Therefore, targeting ß-catenin is an attractive therapeutic strategy for cancers. METHODS: Herein, we identified a natural, small molecule inhibitor of ß-catenin signaling, BASI, and evaluated its therapeutic efficacy both in vitro and in orthotopic mouse models of glioma. RESULTS: BASI significantly suppressed proliferation and invasion and induced apoptosis in glioblastoma cells and resulted in the remarkable attenuation of orthotopic tumor growth in vivo. Furthermore, we found that BASI altered the expression of several microRNAs, which mediated the posttranscriptional silencing of ß-catenin expression either directly or indirectly through a von Hippel-Lindau (VHL)-mediated ß-catenin degradation pattern. CONCLUSIONS: Taken together, our findings offer preclinical validation of BASI as a promising new type of ß-catenin inhibitor with a mechanism of inhibition that has broad potential for the improved treatment of glioblastoma.

Prevention of cancer and inflammation by soybean protease inhibitors.

Several plant-based nutrients and non-nutrients that can inhibit mutagenesis and cell proliferation have been identified. Some of the most promising compounds identified as chemopreventive and anti-metastatic agents include soybean-derived protease inhibitors (PIs), Bowman-Birk Inhibitor (BBI) and Kunitz-Trypsin Inhibitor (KTI). A growing body of evidence suggests that BBI could act as anti-carcinogenic agent and KTI is considered to prevent cancer invasion and metastasis. These inhibitors are non-toxic, are of low cost and can be taken orally or as a part of the daily diet. PIs are undergoing investigation in the clinical setting as potential agents for chemoprevention and anti-metastasis. A complex scenario about the interaction between PIs and cell signaling has been emerging. Soybean PIs are not just anti-proteolytic proteins, but can also be modulators of cell signal transduction. Cancer and inflammatory treatment strategies modulating signal transduction need further investigation.

The impaired viability of prostate cancer cell lines by the recombinant plant kallikrein inhibitor.

BACKGROUND: Kallikreins play a pivotal role in establishing prostate cancer. RESULTS: In contrast to the classical Kunitz plant inhibitor SbTI, the recombinant kallikrein inhibitor (rBbKIm) led to prostate cancer cell death, whereas fibroblast viability was not affected. CONCLUSION: rBbKIm shows selective cytotoxic effect and angiogenesis inhibition against prostate cancer cells. SIGNIFICANCE: New actions of rBbKIm may contribute to understanding the mechanisms of prostate cancer. Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145.

Anti-angiogenic effect of soybean kunitz trypsin inhibitor on human umbilical vein endothelial cells.

Soybean kunitz trypsin inhibitor (STI) was purified from aqueous extract of defatted soybean meal by affinity and ion exchange chromatography. In this study the effect of purified STI on cell migration and tubulogenesis in microcarrier-based fibrin gel was assayed. Purified STI had strong inhibitory effect on human umbilical vein endothelial cells migration and tubulogenesis in fibrin matrix, without toxic effects in the studied doses.

Peltophorum dubium and soybean Kunitz-type trypsin inhibitors induce human Jurkat cell apoptosis.

Plants constitute an important source of compounds which can induce apoptosis in a variety of cells. Previously, we reported the isolation of a trypsin inhibitor from Peltophorum dubium seeds (PDTI). This inhibitor, as well as soybean trypsin inhibitor (SBTI), both belonging to the Kunitz family, have lectin-like properties and trigger rat lymphoma cell apoptosis. In the present study, we demonstrate for the first time that PDTI and SBTI induce human leukemia Jurkat cell death. Induction of apoptosis was confirmed by flow cytometry after propidium iodide labeling of apoptotic nuclei, showing a considerable increase of the sub G(0)/G(1) fraction, with no cell cycle arrest. With the purpose of gaining insight into the signaling pathways involved, we investigated the activation of caspases and the effect of caspase inhibitors, and showed caspases-3 and -8-like activation by PDTI or SBTI-treatment. Consistent with these results, pan caspase inhibitor and caspase-8 inhibitor protected Jurkat cells from apoptosis. However, there was no caspase-9 activation, confirmed by the failure of caspase-9 inhibitor to prevent cell death. No significant release of cytochrome c from mitochondria was detected suggesting that the intrinsic mitochondrial pathway is not predominant in the apoptotic process. On the other hand, recruitment of Fas-associated death domain (FADD) to the cell membrane indicates the involvement of this adaptor protein in PDTI- and SBTI-induced apoptosis in Jurkat cells. Furthermore, human peripheral lymphocytes, either stimulated with phytohemagglutinin or not, are also susceptible to viability decrease induced by these inhibitors.

Hepatocyte growth factor activation inhibitors (HAI-1 and HAI-2) regulate HGF-induced invasion of human breast cancer cells.

Hepatocyte growth factor (HGF) plays a plethora of roles in cancer metastasis and tumour growth. The interaction between tumour cells and their surrounding stromal environment is a crucial factor regulating tumour invasion and metastasis. Stromal fibroblasts are the main source of HGF in the body, and release HGF as an inactive precursor (pro-HGF). HGF activator (HGFA), matriptase, urokinase-type plasminogen activator and hepsin are the main factors responsible for converting pro-HGF into active HGF. HAI-1 and HAI-2 are 2 novel Kunitz-type serine protease inhibitors that regulate HGF activity through inhibition of HGFA, matriptase and hepsin action. Recent studies demonstrate that HAI-1 and HAI-2 may also potently inhibit a number of other pro-metastatic serine proteases and therefore have direct bearing on the spread of tumours. Our study examined the potential of these HAI's to suppress the influence of HGF and regulate cancer metastasis. We generated a retroviral expression system that induced HAI expression in a human fibroblast cell line. Forced expression of either HAI-1 or HAI-2 in these fibroblasts resulted in a dramatic decrease in the production of bioactive hepatocyte growth factor (HGF). This reduction in HGF activity subsequently suppressed HGF's metastatic influence on breast cancer cells. To further assess the anti-cancer properties of HAI-1 and HAI-2 we generated recombinant HAI proteins. These recombinant HAI proteins possessed the ability to potently quench HGF activity. We also demonstrate that these recombinant HAI's suppressed fibroblast-mediated breast cancer invasion. An additional ribozyme transgenes study revealed that elimination of HAI-1 and HAI-2 expression, in an MDA-MB-231 breast cancer cell line, significantly enhanced the migratory, proliferative and invasive nature of these breast cancer cells. Overall, our data demonstrates the important roles of HAI-1 and HAI-2 in cancer metastasis, and reveals that these serine protease inhibitors display strong therapeutic potential.

Suppression of lipopolysaccharide-induced cytokine production of gingival fibroblasts by a soybean, Kunitz trypsin inhibitor.

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.

A soybean Kunitz trypsin inhibitor reduces tumor necrosis factor-alpha production in ultraviolet-exposed primary human keratinocytes.

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.

Suppression of urokinase expression and invasion by a soybean Kunitz trypsin inhibitor are mediated through inhibition of Src-dependent signaling pathways.

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.

Dietary supplementation of soybean kunitz trypsin inhibitor reduces lipopolysaccharide-induced lethality in mouse model.

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.

Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2.

Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer and promotes tumor progression and metastasis. We generated a soluble form of hepsin comprising the entire extracellular domain to show that it efficiently converts single-chain hepatocyte growth factor (pro-HGF) into biologically active two-chain HGF. Hepsin activity was potently inhibited by soluble forms of the bi-Kunitz domain inhibitors HAI-1B (IC(50) 21.1+/-2.7 nM) and HAI-2 (IC(50) 1.3+/-0.3 nM). Enzymatic assays with HAI-1B Kunitz domain mutants (R260A and K401A) further demonstrated that inhibition was due to Kunitz domain-1. The results suggest a functional link between hepsin and the HGF/Met pathway, which may contribute to tumor progression.

Bikunin suppresses lipopolysaccharide-induced lethality through down-regulation of tumor necrosis factor- alpha and interleukin-1 beta in macrophages.

BACKGROUND: Lipopolysaccharide (LPS) is the primary mediator of gram-negative sepsis; it induces the production of macrophage-derived cytokines. It has been shown that bikunin, a Kunitz-type protease inhibitor, inhibits LPS-induced cytokine expression. METHODS: To explore the role of bikunin, bikunin knockout (Bik(-/-)) mice were used for in vitro cytokine experiments and in vivo animal models. RESULTS: We show that a higher level of LPS-mediated death was induced in Bik(-/-), compared with wild-type (wt), mice; the administration of bikunin caused a significant reduction in LPS-induced lethality; LPS significantly increased tumor necrosis factor (TNF)- alpha and interleukin-1 beta levels in Bik(-/-), relative to wt, mice after LPS challenge; concomitant administration of bikunin inhibited the LPS-induced plasma levels of these cytokines; bikunin suppressed the LPS-induced up-regulation of cytokine expression through the suppression of the phosphorylation of ERK1/2, JNK, and p38 in macrophages; and LPS-induced up-regulation of TNF- alpha expression was not enhanced in Bik(-/-) macrophages without endogenous bikunin. CONCLUSIONS: These data allow us to speculate that the increased sensitivity of Bik(-/-) mice to LPS-induced death in vivo is due to a lack of circulating bikunin in plasma. Bikunin may play a role as a potent anti-inflammatory agent.

Bikunin down-regulates heterodimerization between CD44 and growth factor receptors and subsequently suppresses agonist-mediated signaling.

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.

A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-beta and subsequently suppresses TGF-beta signalings.

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.

Suppressing effects of dietary supplementation of soybean trypsin inhibitor on spontaneous, experimental and peritoneal disseminated metastasis in mouse model.

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.

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 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.

Therapeutic efficacy of once-daily oral administration of a Kunitz-type protease inhibitor, bikunin, in a mouse model and in human cancer.

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.

Genetic downregulation of pregnancy-associated plasma protein-A (PAPP-A) by bikunin reduces IGF-I-dependent Akt and ERK1/2 activation and subsequently reduces ovarian cancer cell growth, invasion and metastasis.

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.

The protease inhibitor bikunin, a novel anti-metastatic agent.

Bikunin is a Kunitz-type protease inhibitor predominantly found in human amniotic fluid. In cancers, administration of bikunin may block tumor cell invasion by a direct inhibition of tumor cell-associated plasmin activity as well as by inhibiting urokinase-type plasminogen activator (uPA) expression at the gene and protein levels, possibly through suppression of CD44 dimerization and/or the MAP kinase signaling cascade. Treatment of cancer patients with bikunin may be beneficial in the adjuvant setting to delay the onset of metastasis development and/or in combination with cytotoxic agents to improve treatment efficacy in patients with advanced ovarian cancer.