Clinical trials utilizing a protein extract of soybean enriched in Bowman-Birk inhibitors (BBIC)
1. Introduction
Serine proteases have long been recognized as major players in a wide range of biological processes including cell signaling, cell cycle progression, digestion, immune responses, blood coagulation and wound healing. Their role in the physiology of many human diseases, ranging from cancer and inflammatory disorders to degenerative diseases, now represents an increasingly important feature of this family of enzymes. Proteases are tightly controlled through a number of different mechanisms, including regulation of gene expression, recognition of the substrate by the active site, activity regulation by small molecules, changes in cellular location, post-translational modifications, interaction with other proteins and/or through inhibition of proteolysis by protease inhibitors (PI) [1-3]. This last mechanism usually involves competition with substrates for access to the active site of the enzyme and the formation of tight inhibitory complexes. An understanding of the role played by serine proteases and their specific inhibitors in human diseases offers novel and challenging opportunities for preventive and/or therapeutic intervention [4].
Within this framework, there is a growing interest in naturally-occurring serine protease inhibitors of the Bowman-Birk family due to their potential chemopreventive and/or therapeutic properties which can impact on several human diseases, including cancer, neurodegenerative diseases and inflammatory disorders. In light of the Food and Drug Administration (FDA) approval for trials of Bowman-Birk inhibitors (BBI) concentrate (BBIC), a protein extract of soybean (
Disease | Type of trial | Experimental design | Main results | Ref. |
Benign prostatic hyperplasia | Phase I trial | Duration: 6 months. 19 patients. Daily doses up to 800 CIU a | Significant decrease (up to 43 %) in PSAb levels after treatment. Decrease of prostate volume in patients. No dose-limiting toxicity | [5] |
Oral leukoplakia | Phase I trial | Duration: 1 month. 24 patients. Single daily dose: 800 CIU | BBI was well tolerated. No clinical evidence of toxicity or any adverse reaction | [6] |
Plase II trial | Duration: 1 month. 32 patients. Administration: twice daily, up to 1066 CIU | 31 % of patients achieved clinical response and lesion area decreased after treatment. Dose-dependent effect. BBI was non-toxic. The positive clinical effect of BBIC could be due to the inhibition of serine proteases involved in the cleavage of neu-oncogen protein on the cell surface, preventing the release of the extracellular domain of the protein into the bloodstream | [7] | |
Double-blindrandomized,Placebo-controlled phase IIb trial | 148 patients. Daily dose: 600 CIU | Although this study has not been completed yet, preliminary results suggest that BBIC is not fully effective in patients | [8] | |
Ulcerative colitis | A randomized, double blind, placebo-controlled trial | 12 weeks of therapy. 28 patients. Daily dose: 800 CIU | BBIC might be associated with the regression of disease without apparent toxicity or adverse side effects | [9] |
Table 1.
2. The Bowman-Birk family
2.1. Sources and occurrence
Plant PI can be categorized into at least 12 different families with 10 of these targeting serine proteases and adopting the standard mechanism of inhibition [10]. Members of the Bowman-Birk family are canonical serine PI of low molecular weight, being particularly abundant in legume seeds. Soybean BBI represent the most extensively studied members of the Bowman-Birk family, but related BBI from other dicotyledonous legumes [including chickpea (
The BBI that are expressed in seeds are the products of multi-gene families. Several isoinhibitors have been identified in seeds of individual species [11, 12]. The expression of distinct genes, together with the post-translational modifications of primary gene products, combine to give rise to the array of BBI-like variants described for many legume species. Variants in overall and active site sequences, size, functional properties and spatial pattern of expression have been described [13]. As a result, qualitative and quantitative differences in protease inhibitory activities have been shown in comparisons of pea genotypes [14, 15]. The close linkage of the genes encoding BBI, demonstrated for a number of legume species [16], allows the development of near-isolines having distinct haplotypes. In pea, the co-segregation of quantitative and qualitative variation has been used to develop a series of near-isolines, which have allowed the biological significance of a five-fold variation in seed protease inhibitory activity to be investigated at the level of ileal digestibility [17, 18]. These lines now facilitate related studies on the positive contribution of seed BBI to the prevention of disease states.
The occurrence of BBI in soy foods (soymilk, soy infant formula, defatted soy meal, oilcake, tofu, soybean protein isolate and soybean protein concentrate, among others) is noteworthy, where BBI may be present in different amounts. The soy varieties used, the products themselves and the technological processes used in their preparations all contribute to variation in BBI concentration. In order to quantify BBI in soy foods, enzymatic and immunological methods have been developed; however, no comprehensive information on the concentration of BBI in soy foods is available currently. Recently, Hernández-Ledesma
2.2. Inhibitory properties
The inhibitory activity of BBI is due to the formation of stable complexes between the inhibitor and target proteases. The conformation of the reactive site loop is complementary to the active site of the protease inhibited and allows BBI to bind tightly to proteases in a substrate-like manner [20, 21]; the resulting non-covalent complex renders the target protease inactive. Upon complex formation, BBI may be cleaved very slowly (low
3. Bioavailability and metabolism of BBI
In order to exert any local or systemic health benefits, dietary BBI must resist degradation and maintain biological activity, at least to some extent, after food processing and further passage through the gastrointestinal tract (GIT) [22]. BBI from several legume sources have been shown to resist thermal treatment (up to 100 °C), under either neutral or acidic conditions [32]. Most of the heat-resistant trypsin inhibitory activity in processed legumes is attributable to BBI. At temperatures of 80 °C or lower, chickpea BBI were found to be stable and their inhibitory activities to be unaffected by thermal treatment [33]. Soybean BBI do not lose activity at pH values as low as 1.5 in the presence of pepsin at 37 °C for 2 h [34]; these proteins are also stable to both the acidic conditions and the action of digestive enzymes under simulated gastric and intestinal digestion [35]. Such stability is associated with the rigid structure provided by the seven intra-molecular disulphide bridges that maintain the structural and functional features of the binding sites by adding covalent attachment to the inhibitor core [10, 36]. BBI are fully inactivated by autoclaving or reduction of their disulphide bridges followed by alkylation of the cysteinyl sulfhydryl groups [26].
The resistance of BBI to extreme conditions within the GIT may favour the transport of biologically active BBI across the gut epithelium and could allow their distribution to target organs or tissues in order to exert their beneficial effects locally. The uptake and distribution of soybean BBI, following oral administration, has been examined in rodents. By using [125I] BBI, it was demonstrated that BBI becomes widely distributed in mice 3 h after oral administration [37]. Labelled BBI was detected in the luminal contents of the small and large intestine; analysis of tissue homogenates revealed also the presence of active BBI in internal organs where soybean BBI have been shown to exert anti-carcinogenic effects (see next section). By using inverted sacs from different sections of the small intestine, it was demonstrated that active BBI could be transported effectively across the gut epithelium. It has been shown that soybean BBI have a serum half-life of 10 h in rats and hamsters, and are excreted in urine and faeces [38]. In humans, BBI are taken up rapidly and can be detected in the urine within 24-48 h [6]. These findings suggest that BBI are absorbed after oral administration and can reach several tissues and organs.
BBI have potential health-promoting properties within the GIT [22].
4. Chemopreventive properties of Bowman-Birk inhibitors
Chemoprevention is the use of natural agents or synthetic drugs to halt or reverse the carcinogenesis process before the emergence of invasive cancer. The fact that certain dietary constituents can exert chemopreventive properties has major public health implications and the widespread, long-term use of such compounds should be promoted in populations at normal risk, based on understanding the scientific basis of their beneficial effects.In particular, BBI have been linked to a possible protective effect against both inflammatory disorders and cancer development (Table 2).
4.1. Colorectal cancer
Nutritional intervention and/or dietary manipulation have been suggested as key strategies to prevent and/or control colorectal carcinogenesis [42, 43], one of the major causes of cancer-related mortality worldwide in both men and women [44]. There is now robust preclinical evidence to suggest that dietary BBI from several legume sources can prevent or suppress cancer development and associated inflammatory disorders within the GIT [22]. Soybean BBI have been reported to be effective at concentrations as low as 10 mg/100 g diet, in reducing the incidence and frequency of colorectal tumors, in studies based on the dimethylhydrazine (DMH) rat model, where no adverse effect of BBI was documented for animal growth or organ physiology [45]. When the inhibitory activity of BBI is abolished, any suppressive effect on colorectal tumor development disappears, suggesting that the inhibitory properties of BBI against serine proteases may be required for their reported chemopreventive properties. Proteases play a critical role in tumorigenesis, where their activities become dysregulated in colorectal cancer and neoplastic polyps [46]. In particular, serine proteases are key players in several biological functions linked to tumor development, including cell growth (dys)regulation and cell invasion as well as angiogenesis and inflammatory disorders. Some of these proteases have been reported as promising cancer biomarkers [47-49] (Table 3). An understanding of the role played by specific serine proteases in the biological processes associated with disease may suggest modes of therapeutic intervention [1, 50]. Successful examples of therapeutic intervention using PI include ubiquitin-proteasome inhibitors in the treatment of multiple myeloma [51]. The ubiquitin-proteasome pathway is essential for most cellular processes, including protein quality control, cell cycle, transcription, signalling pathways, protein transport, DNA repair and stress responses [52]. Inhibition of proteasome activity leads to accumulation of poly-ubiquitinylated and misfolded proteins, endoplastic reticulum stress and eventually apoptosis. Although soybean BBI has been demonstrated to inhibit the proteasomal activity of MCF7 breast cancer cells (see section 4.4), the proteasomal inhibition in colon cancer cells need to be unambiguously demonstrated. Another potential therapeutic target of BBI is matriptase (also known as MT-SP1 or epithin), an epithelial-specific member of the type II transmembrane serine protease family, which plays a critical role in differentiation and function of the epidermis, gastrointestinal epithelium and other epithelial tissues. Several studies suggest that matriptase is over-expressed in a wide variety of malignant tumors including prostate, ovarian, uterine, colon, epithelial-type mesothelioma and cervical cell carcinoma [53]. It has been proposed to have multiple functions, acting as a potential activator of critical molecules associated with tumor invasion and metastasis. MT-SP1 contributes to the upstream activation of tumor growth and its progression through the selective degradation of extracellular matrix proteins and activation of cellular regulatory proteins, such as urokinase-type plasminogen activator, hepatocyte-growth factor/scatter factor and protease-activated receptor [54]. Although the ability of soybean BBI to inhibit a secreted form of recombinant MT-SP1 has been demonstrated [55], the clinical relevance of such inhibition has not been proven yet. The validation of specific serine proteases as BBI targets, together with the identification of natural BBI variants, and the design of specific potent inhibitors of these proteases, will contribute to the assessment of BBI as colorectal chemopreventive agents for preventive and/or therapeutic medicine [22].
Colorectal | Soybean | DMHa | Colon carcinogenesis in rodents | Reduction of incidence and frequency of tumors likely via protease inhibition | [45] |
Soybean | DMH | Mouse colon and anal inflammation | Suppression of adenomatous tumors of the GIT | [56] | |
Soybean | DSSb | Mouse colon inflammation | Suppression of histological inflammation parameters, lower mortality rate and delayed onset of mortality | [57] | |
Horsegram | DMH | Colorectal carcinogenesis | Protective role against inflammation and pre-neoplastic lesions | [58] | |
Lentil | - | Colon cancer cells | Proliferation of HT29 colon cancer cells was decreased (IC50 = 32 µM), whereas the non- malignant fibroblastic CCD18Co cells were unaffected | [30] | |
Pea | - | Colon cancer cells | The anti-proliferative effect of BBI in colon cancer cells are demonstrated | [15] | |
Soybean | - | Colon cancer cells | Time- and concentration-dependent anti-proliferative effect on HT29 cells, arrest at G0-G1 phase; trypsin- and chymotrypsin-like proteases are potential targets | [26] | |
Recombi-nant proteins | - | Colon cancer cells | rTI1B, a major BBI isoinhibitor from pea, having trypsin and chymotrypsin inhibitory activity, affected the proliferation of colon cancer cells; however, a derived inactive mutant did not show any anti-proliferative effect | [68] | |
Gastric | Field bean | Benzo-pyrene | Mouse stomach carcinogenesis | BBI was more effective in prevention than in therapeutic treatment, with activity related to its protease inhibitory ability | [100] |
Breast | Black-eyed pea | - | Breast cancer cells | BBI induced apoptosis, cell death andlysosome membrane permeabilization | [79] |
Soybean | - | Breast cancer cells | Proteasome was reported as potential therapeutic target in MCF-7 cells | [78] | |
Prostate | Soybean | - | Prostate cancer cells and rat prostate carcinogenesis | BBI exerted chemopreventive activity associated with induction of connexin-43 expression and apoptosis | [76,77] |
Soybean | - | Prostate cancer xeno-grafts in nude mice | BBI and BBIC inhibited the growth of LNCaP cells | [72] | |
Soybean | - | Prostate cancer cells | BBI prevented the generation of activated oxygen species and activated DNA repair through a p53-dependent mechanism | [74, 75] | |
Oral | Soybean | - | Oral leukoplakia | BBIC exerted a dose-dependent reduction in oral lesion size in 31% of patients without any adverse effects; modulation of protease activity and | [6,7,69] |
Table 2.
A strong interest exists in investigating the potential of BBI as anti-inflammatory agents within the GIT. In rodents, soybean BBI treatment appears to have a potent suppressive effect on colon and anal gland inflammation, following exposure to carcinogenic agents [56], or when assessed in an acute injury/colitis model [57].The protective effect of BBI from soybean or those from perennial horsegram(
Tryptase | Phagocytosis, degradation of ECMa compounds, regulation of inflammatory responses, blood coagulation | Atherosclerosis, asthma, inflammatory disorders | [97, 98] |
Cathepsin G | ECM degradation, migration, regulation of inflammatory disorders | Inflammation, metastasis | [62] |
Matriptase | Matrix degradation, regulation of intestinal barrier, iron metabolism | Pathogenesis of epithelial tissues, tumor growth and progression | [55] |
Human elastase | Pathogen killing, ECM degradation, inflammatory disorders | Pulmonary disease, inflammation | [62, 99] |
Chymase | Degradation of ECM compounds, regulation of inflammatory responses | Inflammation, asthma, gastric cancer | [64] |
Proteasome | Protein degradation, cell proliferation, differentiation, angiogenesis and apoptosis | Carcinogenesis, inflammation, neurodeg-eneration | [58, 78] |
Table 3.
Serine proteases involved in pathological processes as potential therapeutic targets of soybean BBI and related proteins (adapted from Clemente et al., 2011 [22]).
In previous studies, a significant concentration- and time-dependent decrease in the growth of an array of colon cancer cells (HT29, Caco2, LoVo) has been demonstrated

Figure 1.
Dose–response effects of rTI1B (closed bars), a major pea isoinhibitor expressed in
4.2. Oral leukoplakia
Leukoplakia in the oral cavity is considered a suitable model for the study of chemoprevention because the precancerous lesions are readily accessible to visual examination, diagnostic sampling and evaluation of response to treatment. In a Phase I clinical trial, no clinical evidence of toxicity or any adverse effect was apparent when BBIC was administered as a single oral dose of up to 800 CIU to twenty-four patients with oral leukoplakia over one month-period [6]. The study revealed that BBIC was well-tolerated and no allergic reactions, gastrointestinal side-effects or other clinical symptoms were elicited. In a non-randomized phase IIa clinical trial, treatment with BBIC for one month resulted in a dose-dependent reduction in oral lesion size in 31% of patients [7]. The positive clinical effect of BBIC was associated with modulation of protease activity and
4.3. Prostate cancer
Prostate cancer is the second most frequently diagnosed cancer in men although the incidence of cancer varies greatly throughout the world. Dietary habits and lifestyle have been identified as major risk factors in prostate cancer growth and progression, suggesting that prostate cancer might be preventable [70]. Epidemiological studies have shown an inverse association between soy intake and the risk of developing prostate cancer[71]. Preclinical and clinical studies have shown the potential chemopreventive properties of BBI in prostate cancer. Purified soybean BBI and BBIChave been shown to inhibit the growth of LNCaP human prostate cancer xenografts in nude mice [72], and to decrease the growth, invasion and clonogenic survival of several human prostate cancer cells [73]. The effectiveness of soybean BBI in preventing the generation of activated oxygen species in prostate cancer cells [74] and in activating DNA repair through a p53-dependent mechanism has been reported [75]. More recently, BBIC has prevented the growth of prostate tumors in transgenic rats developing adenocarcinoma, most likely as a consequence of its anti-proliferative activity via induction of connexin 43 expression [76,77]. In humans, a double-blind, randomized, phase I trial was carried out in nineteen male subjects with benign prostatic hyperplasia, which is a precursor condition for prostate cancer, and lower urinary tract symptoms[5]. In this study, the authors demonstrated that BBIC treatment for six months reduced levels of prostate-specific antigen (PSA), a clinical marker for prostate cancer, and prostate volume in patients. Additional clinical studies will be necessary to determine the potential of BBIC as prostate cancer chemopreventive agent.
4.4. Breast cancer
Breast cancer is one of the most frequent cancer types and is responsible for the highest mortality rate among women. Novel complementary strategies, including chemoprevention, have been suggested. As 125I-BBI, when orally administrated in rodents, has been demonstrated in the bloodstream and distributed through the body [37-38], its chemopreventive properties could occur in breast tissue.
4.5. Radioprotection
Radiotherapy is used in the treatment of a broad range of malignant tumors with the aim to inflict maximal damage on the tumor tissue. Exposure of surrounding normal tissue to therapeutic radiation should be minimized to avoid side effects that can have a significant impact on general status and quality of life of patients. The use of radioprotective agents to reduce the damage in normal tissue may improve the therapeutic benefit of radiotherapy. The radioprotective properties of BBI have been tested on cell cultures; so far, no data regarding efficacy in humans are available. Soybean BBI have shown potent and selective radioprotection of normal tissue
The involvement of BBI in radiation-induced signaling cascades, and their role in stabilizing a specific tyrosine phosphatase that interferes with the activation of an epidermal growth factor receptor in response to radiation exposure, could be responsible for such protection [84]. Experiments carried out with linear forms of BBI demonstrated that the secondary structure of BBI, required for the protease inhibitory activity, was not necessary for its radioprotective properties [85]. The radioprotective effect of soybean BBI was mainly associated with the chymotrypsin inhibitory site [86] and could be mimicked using a synthetic linearized nonapeptide (CALSYPAQC), corresponding to the active site for chymotrypsin inhibition, but lacking protease inhibitor activity [85]. These observations provide opportunities for the use of synthetic peptides for protecting against ionizing radiation. BBI, when applied topically, once a day for 5 days, to SKH-1 hairless mice with a high risk of developing UV-induced skin tumors, inhibited the formation and growth of skin tumors [85]. In addition, topical application of nondenatured soymilk, once a day for a period of five days prior to UV irradiation, to mini-swine skin reduced or completely eliminated UV-induced formation of thymine dimers and apoptotic cells. Finally, BBIC appears to play a radioprotective role in radiation-induced cataract formation reducing the prevalence and severity of the lens opacifications in mice exposed to high-energy protons [88].
5. Beneficial properties of Bowman-Birk inhibitors in non-related cancer diseases
The loss of muscle protein due to inactivity, disease or aging is a process known as muscular atrophy or wasting. Skeletal muscular atrophy in response to disuse involves both a decrease in protein synthesis and increased protein degradation, predisposing humans to undergo a substantial loss of muscle mass. In connection with this, complex proteolytic cascades may provide a mechanism for the initiation of protein degradation during atrophy. Dietary intervention suggests possible therapeutic strategies
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by progressive demyelination of the brain and spinal cord. Available therapeutic treatments have only limited efficacy and show significant side effects. The search for novel therapeutic agents that can be administered orally, and act synergistically with existing therapies, would be useful for patients with MS. Purified soybean BBI and BBIC have been shown to be effective in the suppression of experimental autoimmune encephalomyelitis in rodents, a model to study the pathogenic mechanisms of MS and to test potential therapies [92]. The oral administration of BBI in mice caused an improvement of several disease parameters (onset, severity, weight loss, inflammation, neuronal loss and demyelination), with no apparent adverse effects [93,94]. Interestingly, BBI ameliorated disease, even when treatment was initiated after disease onset,
6. Concluding remarks
In recent years, much effort has focused on clarifying the potential chemopreventive properties of BBI. Preclinical and clinical studies have clearly demonstrated that BBI uptake is well-tolerated and no side-effects were elicited. This is particularly relevant and lack of toxicity is a major consideration, given the necessity for prolonged duration of administration. Consistently, several studies have shown that serine proteases are potential BBI targets in prevention and therapy; however, these targets have not been proven thus far. The validation of specific serine proteases as BBI targets will contribute to the assessment of BBI as chemopreventive agents that may be used in preventive and/or therapeutic medicine.
Acknowledgement
A.C. acknowledges support byERDF-co-financed grantsfrom the Spanish CICYT (AGL2010-15877AGL and AGL2011-26353). A.C. is involved in COST Action FA1005 INFOGEST on Food Digestion. C.D. acknowledges support from the European Union (Grain Legumes Integrated Project, a Framework Programme 6 project, grant no. FOOD-CT-2004-506223) and from Defra, United Kingdom (grant nos. AR0105 and AR0711).
References
- 1.
Nature Reviews Drug DiscoveryTurk B. Targeting Proteases. Successes Failures. Future Prospects. 2006 5 785 799 - 2.
Allosteric Regulation of Protease Activity by Small Molecules. Molecular BiosystemsShen A. 2010 6 1431 1443 - 3.
Emerging Principles in Protease-based Drug Discovery. Nature Reviews Drug DiscoveryDrag M. Salvesen G. S. 2010 9 690 701 - 4.
New Approaches for Dissecting Protease Functions to Improve Probe Development and Drug Discovery. Nature Structural & Molecular BiologyDeu E. Verdoes M. Bogyo M. 2012 19 9 16 - 5.
ProstateMalkowicz S. B. Mc Kenna W. G. Vaughn D. J. Wan X. S. Propert K. J. Rockwell K. Marks S. H. F. Wein A. J. Kennedy A. R. Effects of. Bowman-Birk Inhibitor. Concentrate . B. B. I. C. in Patients. with Benign. Prostatic Hyperplasia. 2001 48 16 28 - 6.
Single-dose Administration of Bowman-Birk Inhibitor Concentrate in Patients with Oral Leukoplakia. Cancer Epidemiology, Biomarkers & PreventionArmstrong W. B. Kennedy A. R. Wan X. S. Atiba J. CE Mc Laren Meyskens. F. L. 2000 9 43 47 - 7.
Clinical Modulation of Oral Leukoplakia and Protease Activity by Bowman-Birk Inhibitor Concentrate in a Phase IIa Chemoprevention Trial. Clinical Cancer ResearchArmstrong W. B. Kennedy A. R. Wan X. S. Taylor T. H. Nguyen Q. A. Jensen J. Thompson W. 2000 6 4684 4691 - 8.
Phase IIb Randomized Clinical Chemoprevention Trial of a Soybean-derived Compound (Bowman-Birk Inhibitor Concentrate) for Oral Leukoplakia. Cancer Prevention ResearchMeyskens F. L. Taylor T. Armstrong W. Kong L. Gu M. Gonzalez R. Villa M. Wong V. Garcia A. Perloff M. Kennedy A. Wan S. Ware J. H. Messadi D. Lorch J. Wirth L. Jaffe Z. Goodwin J. Civantos F. Sullivan M. Reid M. Merciznu M. Jayaprakash V. Kerr A. R. Le A. 2010 CN02 05 - 9.
Digestive Diseases and SciencesLichtenstein G. R. Deren J. Katz S. JD Lewis Kennedy. A. R. Ware J. H. Bowman-Birk Inhibitor. Concentrate A. Novel Therapeutic. Agent for. Patients with. Active Ulcerative. Colitis 2008 53 175 180 - 10.
Bateman KS, James MNG. Plant Proteinase Inhibitors: Structure and Mechanism of Inhibition. Current Protein & Peptide Science2011 12 341 347 - 11.
Three Classes of Proteinase Inhibitor Gene Have Distinct but Overlapping Patterns of Expression in Pisum sativum Plants. Plant Molecular BiologyDomoney C. Welham T. Ellis N. Mozzanega P. Turner L. 2002 48 319 329 - 12.
In silico Characterization and Expression Analysis of the Multigene Family Encoding the Bowman-Birk Protease Inhibitor in Soybean. MolecularBiology ReportsDe Almeida B. Garcia da. Silva W. Alves M. Gonzalves E. 2012 39 327 334 - 13.
Biological Significance of Polymorphism in Legume Protease Inhibitors from the Bowman-Birk Family. Current Protein & Peptide ScienceClemente A. Domoney C. 2006 7 201 216 - 14.
Trypsin Inhibitors in Pisum: Variation in Amount and Pattern of Accumulation in Developing Seed. Seed Science ResearchDomoney C. Welham T. 1992 2 147 154 - 15.
Journal of Agricultural and Food ChemistryClemente A. Gee J. M. Johnson I. T. Domoney C. Pea . Pisum sativum. L. Protease Inhibitors. from the. Bowman-Birk Class. Influence the. Growth of. Human Colorectal. Adenocarcinoma H. T. Cells in. vitro 2005 53 8979 8986 - 16.
Combinatorial Variation in Coding and Promoter Sequences of Genes at the Tri Locus in Pisum sativum Accounts for Variation in Trypsin Inhibitor Activity in Seeds. Molecular Genetics and GenomicsPage D. Aubert G. Duc G. Welham T. Domoney C. 2002 267 359 369 - 17.
The Apparent Ileal Digestibility, Determined with Young Broilers, of Amino Acids in Near-isogenic Lines of Peas (Pisum sativum L.) Differing in Trypsin Inhibitor Activity. Journal of the Science of Food and AgricultureWiseman J. Al-Mazooqi W. Welham T. Domoney C. 2003 83 644 651 - 18.
The Effects of Genetic Variation at r, rb and Tri Loci in Pisum sativum L. on Apparent Ileal Digestibility of Amino Acids in Young Broilers. Journal of the Science of Food and AgricultureWiseman J. Al-Marzooqi W. Hedley C. Wang T. L. Welham T. Domoney C. 2006 86 436 444 - 19.
Lunasin and Bowman-Birk Protease Inhibitor (BBI) in US Commercial Soy Foods. Food ChemistryHernández-Ledesma B. Hsieh C. C. de Lumen B. O. 2009 115 574 580 - 20.
Natural Protein Proteinase-Inhibitors and their Interaction with Proteinases. European Journal of BiochemistryBode W. Huber R. 1992 204 433 451 - 21.
Reactive Sites of an Anticarcinogenic Bowman-Birk Proteinase Inhibitor are Similar to Other Trypsin Inhibitors. The Journal of Biological ChemistryChen P. Rose J. Love R. Wei C. H. Wang B. C. 1992 267 1990 1994 - 22.
Bowman-Birk Inhibitors from Legumes on Human Gastrointestinal Health: Current Status and Perspectives. Current Protein & Peptide ScienceClemente A. Sonnante G. Domoney C. Bowman 2011 12 358 373 - 23.
On the Size of the Active Site in Proteases. I. Papain. Biochemical and Biophysical Research CommunicationsSchechter I. Berger A. 1967 27 157 162 - 24.
Seeds. Molecular BiosystemsRocco M. Marloni L. Chambery A. Poerio E. Parente A. Di Maro A. A. Bowman-Birk Inhibitor. with Anti-elastase. Activity from. Lathyrus sativus. L. 2011 7 2500 2507 - 25.
Polymorphism of Trypsin and Chymotrypsin Binding Loops in Bowman-Birk Inhibitors from Common Bean (Phaseolus vulgaris L.). Plant SciencePiergiovanni A. R. Galasso I. 2004 166 1525 1531 - 26.
The Cytotoxic Effect of Bowman-Birk Isoinhibitors, IBB1 and IBBD2, from Soybean (Glycine max) on HT29 Human Colorectal Cancer Cells is Related to their Intrinsic Ability to Inhibit Serine Proteases. Molecular Nutrition & Food ResearchClemente A. Moreno J. Marín-Manzano M. C. Jiménez E. Domoney C. 2010 54 396 405 - 27.
Proteinase Inhibitors from Pea Seeds: Purification and Characterization. Journal of Agricultural and Food ChemistryFerrasson E. Quillien L. Gueguen J. 1997 45 127 131 - 28.
The Effect of Variation within Inhibitory Domains on the Activity of Pea Protease Inhibitors from the Bowman-Birk Class. Protein Expression and PurificationClemente A. Mac Kenzie. D. A. Jeenes D. J. Domoney C. 2004 36 106 114 - 29.
Inhibitory Properties and Solution Structure of a Potent Bowman-Birk Protease Inhibitor from Lentil (Lens culinaris, L.) Seeds. FEBS JournalRagg E. M. Galbusera V. Scarafoni A. Negri A. Tedeschi G. Consoni A. Sessa F. Duranti M. 2006 273 4024 4039 - 30.
Bowman-Birk Inhibitors in Lentil: Heterologous Expression, Functional Characterisation and Anti-proliferative Properties in Human Colon Cancer Cells. Food ChemistryCaccialupi P. Ceci L. R. Siciliano R. A. Pignone D. Clemente A. Sonnante G. Bowman 2010 120 1058 1066 - 31.
Identification and Characterization of a Bowman-Birk Inhibitor Active Towards Trypsin but not Chymotrypsin in Lupinus albus Seeds. PhytochemistryScarafoni A. Consonni A. Galbusera V. Negri A. Tedeshi G. Rasmussen P. Magni C. Duranti M. 2008 69 1820 1825 - 32.
Osman MA, Reid PM, Weber CW. Thermal Inactivation of Tepary Bean (Phaseolus acutifolius), Soybean and Lima Bean Protease Inhibitors: Effect of Acidic and Basic pH. Food Chemistry2002 78 419 423 - 33.
Factors Affecting the in vitro Protein Digestibility of Chickpea Albumins. Journal of the Science of Food and AgricultureClemente A. Vioque J. Sánchez-Vioque R. Pedroche J. Bautista J. Millán F. 2000 80 79 84 - 34.
Weder JK. Inhibition of Human Proteinases by Grain Legumes. Advances in Experimental Medicine and Biology1986 199 239 279 - 35.
Park JH, Jeong HJ, Lumen BOD. In Vitro Digestibility of the Cancer-Preventive Soy Peptides Lunasin and BBI. Journal of Agricultural and Food Chemistry2007 55 10703 10706 - 36.
Trivedi MV, Laurence JS, Siahann TJ. The Role of Thiols and Disulfides on Protein Stability. Current Protein & Peptide Science2009 10 614 625 - 37.
Billings PC, St Clair WH, Maki PA, Kennedy AR. Distribution of the Bowman-Birk Protease Inhibitor in Mice Following Oral Administration. Cancer Letters1992 62 191 197 - 38.
Kennedy AR. Chemopreventive Agents: Protease Inhibitors. Pharmacology & Therapeutics 1998 78 167 209 - 39.
Biological Effects and Survival of Trypsin Inhibitors and the Aglutinin from Soybean in the Small Intestine of the Rat. Journal of Agricultural and Food ChemistryHajós G. Gelencser E. Pustzai A. Grant G. Sakhri M. Bardocz S. 1995 43 165 170 - 40.
Active Bowman-Birk Inhibitors Survive Gastrointestinal Digestion at the Terminal Ileum of Pigs fed Chickpea-Based Diets. Journal of the Science of Food and AgricultureClemente A. Jiménez E. Marín-Manzano M. C. Rubio L. A. 2008 88 513 521 - 41.
Anti-carcinogenic Soyabean Bowman-Birk Inhibitors Survive Fermentation in their Active Form and do not Affect the Microbiota Composition In Vitro. The British Journal of NutritionMarín-Manzano M. C. Ruiz R. Jiménez E. Rubio L. A. Clemente A. 2009 101 967 971 - 42.
Reddy BS. Novel Approaches in the Prevention of Colon Cancer by Nutritional Manipulation and Chemoprevention. Cancer Epidemiology, Biomarkers & Prevention2000 9 239 247 - 43.
Pan MH, Lai CS, Wu JC, Ho CT. Molecular Mechanisms for Chemoprevention of Colorectal Cancer by Natural Dietary Compounds. Molecular Nutrition & Food Research2011 55 32 45 - 44.
Cancer Journal for CliniciansJemal A. Siegel R. Ward E. Hao Y. P. Xu J. Q. MJ Thun Cancer. Statistics 2009 59 225 249 - 45.
Kennedy AR, Billings OC, Wan XS, Newberne PM. Effects of Bowman-Birk Inhibitor on Rat Colon Carcinogenesis. Nutrition and Cancer2002 43 174 186 - 46.
Cancer: Implications for Molecular Detection of Neoplasia. Cancer Epidemiology, Biomarkers & PreventionChan A. T. Baba Y. Sima K. Nosho K. Chung D. C. Hung K. E. Mahmood U. Madden K. Poss K. Ranieri A. Shue D. Kucherlapati R. Fuch C. S. Ogino S. Cathepsin B. Expression Survival in. Colon Cancer. Implications for. Molecular Detection. of Neoplasia. 2010 19 2777 2785 - 47.
Decrease Levels of Secretory Leukoprotease Inhibitor in the Pseudomonas-Infected Cystic Fibrosis Lung are Due to Neutrophil Elastase Degradation. Journal of ImmunologyWeldon S. Mc Nally P. Mc Elvaney N. G. Elborn J. S. Mc Auley D. F. Wartelle J. Belaaouaj A. Levine R. J. Taggart C. C. 2009 183 8148 8156 - 48.
Clinical Significance of Human Kallikrein7 Gene Expression in Colorectal Cancer. The Annals of Surgical OncologyInoue Y. Yokobori T. Yokoe T. Toiyama Y. Miki C. Mimori K. Mori M. Kusunoki M. 2010 17 3037 3042 - 49.
Evaluation and Prognostic Significance of Human Tissue Kallikrein-related Peptidase 6 (KLK6) in Colorectal Cancer. Pathology Research and PracticePetraki C. Dubinski W. Scorilas A. Saleh C. MD Pasic Komborozo. V. Khalil B. Gabril M. Y. Streutker C. Diamandis E. P. Yousef G. M. 2012 208 104 108 - 50.
Scott CJ, Taggart CC. Biologic Protease Inhibitors as Novel Therapeutic Agents. Biochimie2010 92 1681 1688 - 51.
Wu W. K. K. Cho C. H. Lee C. W. Wu K. Fan D. Yu J. Sung J. J. Y. Proteasome Inhibition. a. New Therapeutic. Strategy to. Cancer Treatment. Cancer Letters. 2010 293 15 22 - 52.
Proteasome Inhibitors Induce Nucleolar Aggregation of Proteasome Target Proteins and Polyadenylated RNA by Altering Ubiquitin Availability.OncogeneLatonen L. Moore H. M. Bai B. Jaamaa S. Laiho M. 2011 30 790 805 - 53.
Journal of Biological ChemistryBugge T. H. Antalis T. M. Wu Q. Type I. I. Transmembrane Serine. Proteases 2009 284 23177 23181 - 54.
Lee SL, Dickson RB, Lin CY. Activation of Hepatocyte Growth Factor and Urokinase/Plasminogen Activator by Matriptase, an Epithelial Membrane Serine Protease. Journal of Biological Chemistry2000 275 36720 36725 - 55.
Inhibition of Membrane-Type Serine Protease 1/Matriptase by Natural and Synthetic Protease Inhibitors.Journal of Nutritional Science and VitaminologyYamasaki Y. Satomi S. Murai N. Tsuzuki S. Fushiki T. 2003 49 27 32 - 56.
Protease Inhibitor Suppression of Colon and Anal Gland Carcinogenesis Induced by Dimethylhydrazine. CarcinogenesisBillings P. C. Newberne P. Kennedy A. R. 1990 11 1083 1086 - 57.
Bowman-Birk Concentrate Reduces Colon Inflammation in Mice with Dextran Sulphate Sodium-Induced Ulcerative Colitis. Digestive Diseases and SciencesWare H. W. Wan S. Newberne P. Kennedy A. R. Bowman 1999 44 986 990 - 58.
Food and Chemical Toxicologyde Paula A. de Abreu P. Santos K. T. Guerra R. Martins C. Castro-Borges W. Guerra M. H. Bowman-Birk Inhibitors. Proteasome Peptidase. Activities Colorectal Pre-neoplasias. Induced by. 1,2-dimethylhydrazine in. Swiss Mice. 2012 50 1405 1412 - 59.
aminosalicylic Acid Enema in the Treatment of Distal Ulcerative Colitis, Proctosigmoiditis and Proctitis. GastroenterologySutherland L. R. Martin F. Greer S. Robinson M. Greenberger N. Saibil F. Martin T. Sparr J. Prokipchuck E. Borgen L. 1987 92 1894 1898 - 60.
Chymotrypsin-specific Protease Inhibitors Decrease H2O2 Formation by Activated Human Polymorphonuclear Leukocytes. CarcinogenesisFrenkel K. Chranzan K. CA Ryan Wiesner. R. Troll W. 1987 8 1207 1212 - 61.
In vitro Reduction of Peroxidation in UVC Irradiated Cell Cultures by Concurrent Exposure with Bowman-Birk Protease Inhibitor. Teratogenesis, Carcinogenesis and MutagenesisBaturay N. Z. Roque H. 1991 11 195 202 - 62.
Larionova NI, Gladysheva IP, Tikhonova TV, Kazanskaya NF. Inhibition of Cathepsin G and Human Granulocyte Elastase by Multiple Forms of Bowman-Birk Type Soybean Inhibitor. Biochemistry-Moscow1993 58 1046 1052 - 63.
Gladysheva IP, Zamolodchikova TS, Sokolova EA, Larionova NI. Interaction Between Duodenase, a Proteinase with Dual Specificity, and Soybean Inhibitors of Bowman-Birk and Kunitz Type. Biochemistry-Moscow1999 64 1244 1249 - 64.
Soybean Bowman-Birk Protease Inhibitor is a Highly Effective Inhibitor of Human Mast Cell Chymase. Archives of Biochemistry and BiophysicsWare J. H. Wan X. S. Rubin H. Schechter N. M. Kennedy A. R. 1997 344 133 138 - 65.
Chymase is a Potent Chemoattractant for Human Monocytes and Neutrophils. Journal of Leukocyte BiologyTani K. Ogushi K. Kido H. Kawano T. Kumori Y. Kamikura T. Cui P. Sone S. 2000 67 585 589 - 66.
Activation of Human Interstitial Procollagenase through Direct Cleavage of the Leu83- Thr84 Bond by Mast Cell Chymase. Journal of Biological ChemistrySaarinen J. Kalkkinen N. Welgus H. G. Kovanen P. T. 1994 269 18134 18140 - 67.
Rapid and Specific Conversion of Precursor Interleukin 1beta (1L-beta) to an Active IL-1 Species by Human Mast Cell Chymase. Journal of Experimental MedicineMizutani H. Schechter N. M. Lazarus G. Black R. A. Kupper T. S. 1991 174 821 825 - 68.
The Anti-proliferative Effects of TI1B, a Major Bowman-Birk isoinhibitor from Pea (Pisum sativum L), on HT29 Colon Cancer Cells are Mediated Through Protease Inhibition. The British Journal of NutritionClemente A. Marín-Manzano M. C. Jiménez E. Arques M. C. Domoney C. 2012 doi.10.1017/S000711451200075X). - 69.
Wan XS, Meyskens FL, Armstrong WB, Taylor TH, Kennedy AR. Relationship Between Protease Activity and neu Oncogene Expression in Patients with Oral Leukoplakia Treated with the Bowman-Birk Inhibitor.Cancer Epidemiology, Biomarkers & Prevention1999 8 601 608 - 70.
Significance of Chemoprevention for Prostate Cancer Development: Experimental in vivo Approaches to Chemoprevention. Pathology InternationalShirai T. 2008 58 1 6 - 71.
Meta-analysis of Soy Food and Risk of Prostate Cancer in Men. International Journal of CancerYan L. Spitznagel E. L. 2005 117 667 669 - 72.
ProstateWan X. S. Ware J. H. Zhang L. Newberne P. M. Evans S. M. Clark C. L. Kennedy A. R. Treatment with. Soybean-derived Bowman. Birk Inhibitor. Increases Serum. Prostate-specific Antigen. Concentration while. Suppressing Growth. of Human. Prostate Cancer. Xenografts in. Nude Mice. 1999b 41 243 252 - 73.
Kennedy AR, Wan XS. Effects of the Bowman-Birk Inhibitor on Growth, Invasion, and Clonogenic Survival of Human Prostate Epithelial Cells and Prostate Cancer Cells. Prostate2002 50 125 133 - 74.
Sun XY, Donald SP, Phang JM. Testosterone and Prostate Specific Antigen Stimulate Generation of Reactive Oxygen Species in Prostate Cancer Cells. Carcinogenesis2001 22 1775 1780 - 75.
The Radioprotective Effect of BBI Is Associated with the Activation of DNA Repair-Relevant Genes. International Journal of Radiation OncologyDittmann K. Virsik-Kopp P. Mayer C. Rave-Frank M. Rodemann H. P. 2003 79 801 808 - 76.
McCormick DL, Johnson WD, Bosland MC, Lubet RA, Steele VE. Chemoprevention of Rat Prostate Carcinogenesis by Soy Isoflavones and Bowman-Birk Inhibitor. Nutrition and Cancer2007 57 184 193 - 77.
Induction of Apoptosis in the LNCaP Human Prostate Carcinoma Cell Line and Prostate Adenocarcinomas of SV40T Antigen Transgenic Rats by the Bowman-Birk. Pathology InternationalTang M. X. Asamoto M. Ogawa K. Naiki-Ito A. Sato S. Takahashi S. Shirai T. 2009 59 790 796 - 78.
Chen YW, Huang SC, Lin-Shiau SY, Lin JK. Bowman-Birk Inhibitor Abates Proteasome Function and Suppresses the Proliferation of MCF7 Breast Cancer Cells Through Accumulation of MAP Kinase Phosphatase-1. Carcinogenesis2005 26 1296 1305 - 79.
Joanitti GA, Azevedo RB, Freitas SM. Apoptosis and Lysosome Membrane Permeabilization Induction on breast Cancer Cells by an Anticarcinogenic Bowman-Birk Inhibitor from Vigna unguiculata Seeds. Cancer Letters2010 293 73 81 - 80.
Bowman-Birk Proteinase Inhibitor (BBI) Modulates Radiosensitivity and Radiation-Induced Differentiation of Human Fibroblasts in Culture. Radiotherapy and OncologyDittmann K. Löffler H. Bamberg M. Rodemann H. P. Bowman 1995 34 137 143 - 81.
Selective Radioprotection of Normal Tissues by Bowman-Birk Proteinase Inhibitor (BBI) in Mice. Strahlentherapie Und OnkologieDittmann K. Toulany M. Classen J. Heinrich V. Milas L. 2005 181 191 196 - 82.
Dittmann KH,Gueven N,Mayer C,Ohneseit P,Zell P,Begg AC,Rodemann HP. The Presence of Wild-Type TP53 is Necessary for the Radioprotective Effect of the Bowman-Birk Proteinase Inhibitor in Normal Fibroblasts. Radiation Research1998 150 648 655 - 83.
The Radioprotector Bowman-Birk Proteinase Inhibitor Stimulates DNA Repair via Epidermal Growth Factor Receptor Phosphorylation and Nuclear Transport. Radiotherapy and OncologyDittmann K. Mayer C. Kehlbach R. Rodemann H. P. 2008 86 375 382 - 84.
Bowman-Birk Protease Inhibitor Reduces the Radiation-Induced Activation of the EGF Receptor and Induces Tyrosine Phosphatase Activity. International Journal of Radiation OncologyGueven N. Dittmann K. Mayer C. Rodemann H. P. Bowman 1998 73 157 162 - 85.
The Radioprotective Potential of the Bowman-Birk Protease Inhibitor is Independent of its Secondary Structure. Cancer LettersGueven N. Dittmann K. Mayer C. Rodemann H. P. 1998 125 77 82 - 86.
Nanomolar Concentrations of Bowman-Birk Soybean Protease Inhibitor Suppress X-ray Induced Transformation In Vitro.Proceedings of the National Academy of Sciences of the United States of AmericaYavelow J. Collins M. Birk Y. Troll W. Kennedy A. R. 1985 82 5395 5399 - 87.
Inhibitory Effect of Topical Applications of Non-denatured Soymilk on the Formation and Growth of UVB-Induced Skin Tumors. Oncology ResearchHuang M. T. Xie J. G. Lin C. B. Kizoulis M. Seiberg M. Shapiro S. Conney A. 2004 14 387 397 - 88.
Davis JG, Wan XS, Ware JH, Kennedy AR Dietary Supplements Reduce the Cataractogenic Potential of Proton and HZE-Particle Radiation in Mice. Radiation Research2010 173 353 361 - 89.
Morris CA, Morris LD, Kennedy AR, Sweeney HL. Attenuation of Skeletal Muscle Atrophy via Protease Inhibition. Journal of Applied Physiology2005 99 1719 1727 - 90.
Bowman-Birk Inhibitor Concentrate Prevents Atrophy, Weakness, and Oxidative Stress in Soleus Muscle of Hindlimb-Unloaded Mice. Journal of Applied PhysiologyArbogast S. Smith J. Matuszczak Y. Hardin B. J. Moylan J. S. JD Smith Ware. J. Kennedy A. R. Reid M. B. Bowman 2007 102 956 964 - 91.
Bowman-Birk Inhibitor Attenuates Dystrophic Pathology in mdx Mice. Journal of Applied PhysiologyCA Morris Selsby. J. T. Morris L. D. Pendrak K. Sweeney H. L. Bowman 2010 109 1492 1499 - 92.
Journal of Experimental MedicineCruz-Orengo L. Holman D. W. Dorsey D. Zhou L. Zhang P. Wright M. EE Mc Candless Patel. J. R. Luker G. D. Littmann D. R. Rusell J. H. Klein R. S. C. X. C. R. Influences Leukocyte. Entry into. the C. N. S. Parenchyma by. Controlling Abluminal. C. X. C. L. Abundance During. Autoimmunity 2011 208 327 339 - 93.
Multiple Sclerosis. Multiple SclerosisGran B. Tabibzadeh N. Martin A. Ventura E. S. Ware J. H. Zhang G. X. Parr J. L. Kennedy A. R. Rostami A. M. The Protease. Inhibitor-Birk Bowman. inhibitor Suppresses. Experimental Autoimmune. Encephalomyelitis a. Potential Oral. Therapy for. Multiple Sclerosis. 2006 12 688 697 - 94.
Bowman-Birk Inhibitor Suppresses Inflammation and Neuronal Loss in a Mouse Model of Multiple Sclerosis. Journal of the Neurological SciencesTouil T. Ciric B. Ventura E. Shindler K. S. Gran B. Tostami A. Bowman 2008 271 191 202 - 95.
Bowman-Birk Inhibitor Attenuates Experimental Autoimmune Encephalomyelitis by Delaying Infiltration of Inflammatory Cells into the CNS. Immunologic ResearchDai H. Ciric B. Zhang G. X. Rostami A. Bowman 2011 51 145 152 - 96.
Journal of NeuroimmunologyDai H. Ciric B. Zhang G. X. Rostami A. Interleukin- Plays a. Crucial Role. in Suppression. of Experimental. Autoimmune Encephalomyelitis. by-Birk Bowman. Inhibitor 2012 245 1 7 - 97.
Inhibition of Human Beta-Tryptase by Bowman-Birk Inhibitor Derived Peptides: Creation of a New Tri-Functional Inhibitor. Bioorganic & Medicinal ChemistryScarpi D. JD Mc Bride Leatherbarrow. R. J. 2004 12 6045 6052 - 98.
Muricken DG, Gowda LR Molecular Engineering of a Small Trypsin Inhibitor Based on the Binding Loop of Horsegram Seed Bowman-Birk Inhibitor. Journal of Enzyme Inhibition and Medicinal Chemistry2011 26 553 560 - 99.
Seeds. Molecular BiosystemsRocco M. Malorni L. Chambery A. Poerio E. Parente A. Di Maro A. A. Bowman-Birk Inhibitor. with-Elastase Anti. Activity from. Lathyrus sativus. L. 2011 7 2500 2507 - 100.
Fernandes AO, Banerji AP. Inhibition of Benzopyrene-Induced Forestomach Tumors by Field Bean Protease Inhibitor. Carcinogenesis1995 16 1843 1846