Protease Inhibitors from Plants as Therapeutic Agents- A Review.

Plant-based diets are a great source of protease inhibitors (PIs). Two of the most well-known families of PIs are Bowman-Birk inhibitors (BBI) and Kunitz-type inhibitors (KTI). The first group acts mainly on trypsin, chymotrypsin, and elastase; the second is on serine, cysteine, and aspartic proteases. PIs can retard or inhibit the catalytic action of enzymes; therefore, they are considered non-nutritional compounds; nevertheless, animal studies and cell line experiments showed promising results of PIs in treating human illnesses such as obesity, cardiovascular diseases, autoimmune diseases, inflammatory processes, and different types of cancer (gastric, colorectal, breast, and lung cancer). Anticarcinogenic activity's proposed mechanisms of action comprise several inhibitory effects at different molecular levels, i.e., transcription, post-transcription, translation, post-translation, and secretion of cancer cells. This work reviews the potential therapeutic applications of PIs as anticarcinogenic and anti-inflammatory agents in human diseases and the mechanisms by which they exert these effects.

Inhibitory effect of Bowman-Birk protease inhibitor on autophagy in MDAMB231 breast cancer cell line.

BACKGROUND: Autophagy has an essential role in cellular energetic balance, cell cycle, and cell death, so the change in autophagy level is crucial in many human diseases such as cancer. Herbal medicine has been widely used to treat cancer. Bowman-Birk protease inhibitor (BBI), a protease inhibitor extracted from soybean, has antitumorigenic, anti-inflammatory, and anti-angiogenic activities. In this study, we evaluated the effect of BBI on the growth of breast cancer cell line and transcript level of autophagy and apoptosis-related genes. MATERIALS AND METHODS: BBI was purified from soybean by ion-exchange chromatography method. The viability of MDA-MB-231 cells that were treated with BBI was measured by MTT assay, and the transcript level of genes involved in autophagy and apoptosis was measured by real-time-polymerase chain reaction (PCR) technique. RESULTS: The results of BBI purification showed that 100 g of the ethanolic fraction yielded 300-mg BBI with more than 95% purity. MTT results revealed that BBI inhibited the cell growth of MDA-MB-231 cell line in a dose-dependent manner, with an IC50 of 200 µg/mL. The results of real-time reverse transcription-PCR exhibited that BBI altered the expression of Atg5, Beclin1, light chain 3-II, and sequestosome1 and increased the Bax/Bcl2 ratio in MDA-MB-231 cell line. CONCLUSION: According to our results, BBI could inhibit autophagy and induce apoptosis in MDA-MB-231 cell line. Thus, BBI may be used as a therapeutic drug in the treatment of breast cancer whether alone or with chemotherapeutic drugs.

In vivo anti-inflammatory efficacy of the combined Bowman-Birk trypsin inhibitor and genistein isoflavone, two biological compounds from soybean.

Soybean Bowman-Birk protease inhibitor (BBI) and genistein, two biological compounds from soybean, are well-known for their anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was designing a BBI-genistein conjugate and then investigating its protective effect on lipopolysaccharide (LPS)-induced inflammation in BALB/c mice, compared with the effects of combination of BBI and genistein. BBI was purified from soybean and the BBI-genistein conjugate was synthesized. The BALB/c mice were intraperitoneally treated 2 hours before LPS induction. Our results showed that treatment with the combination of BBI and genistein greatly led to more reduced serum levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ compared with the treatments of BBI alone, the BBI-genistein conjugate, and genistein alone, respectively. Moreover, the expression of TNF-α and IFN-γ in the splenocytes was significantly downregulated along with improving host survival against the LPS-induced lethal endotoxemia in the same way. Our data support a new combined therapy using BBI and genistein, as natural anti-inflammatory agents, to develop a new drug for inflammatory diseases.

Oral Cancer Chernoprevention: Current Status and Future Direction.

The aim of this study is to review the current status of cancer chemoprevention and its effectiveness in treatment of oral premalignant lesions and prevention of their progression to oral cancer. The challenges encountered in the different oral cancer chemoprevention clinical trials, including lack of surrogate endpoints, reversal of histologic premalignant changes as study endpoints, tobacco use, human papillomavirus, delivery system, adverse effects and risk of bias in clinical studies, are presented.

Bowman-Birk inhibitor concentrate suppresses experimental autoimmune neuritis via shifting macrophages from M1 to M2 subtype.

BACKGROUND: In the present study, we investigated the immuno-regulatory and therapeutic effects of Bowman-Birk inhibitor concentrate (BBIC) on experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome (GBS) in human. METHODS: EAN in Lewis rats induced by inoculation with peripheral nerve myelin P0 protein peptide 180-199 (P0 peptide) was treated with BBIC at two different therapeutic regimens. RESULTS: Our data indicated that the administration of BBIC daily orally effectively inhibited and ameliorated the clinical and pathological signs of EAN. The suppression of EAN was associated with an insufficiency of autoreactive T cells, as reflected by inhibited P0 peptide-specific mononuclear cell proliferation and decreased in CD4 and CD8T cells infiltrating into the peripheral nervous system (PNS). BBIC might mediate its therapeutic effects by shifting macrophages from M1 to M2 subtype as evidenced by increasing Arg-1, CD206 and IL-10 and inhibiting IFN-γ, TNF-α, IL-12, iNOS and CD40 expressions on macrophages as well as enhancing anti-inflammatory cytokines IL-4 and IL-10 and decreasing inflammatory cytokines, IFN-γ, TNF-α and IL-17 in the PNS. CONCLUSION: Our results suggest that BBIC may have therapeutic potential in human GBS and other autoimmune diseases in the future.

Bowman-Birk inhibitors from legumes as colorectal chemopreventive agents.

Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract (GIT) has prompted scientists to investigate the potential of serine protease inhibitors, both natural and synthetic, as modulators of their proteolytic activities. Protease inhibitors of the Bowman-Birk type, a major protease inhibitor family in legume seeds, which inhibit potently and specifically trypsin- and chymotrypsin-like proteases, are currently being investigated as colorectal chemopreventive agents. Physiologically relevant amounts of Bowman-Birk inhibitors (BBI) can reach the large intestine in active form due to their extraordinary resistance to extreme conditions within the GIT. Studies in animal models have proven that dietary BBI from several legume sources, including soybean, pea, lentil and chickpea, can prevent or suppress carcinogenic and inflammatory processes within the GIT. Although the therapeutic targets and the action mechanism of BBI have not yet been elucidated, the emerging evidence suggests that BBI exert their preventive properties via protease inhibition; in this sense, serine proteases should be considered as primary targets in early stages of carcinogenesis. The validation of candidate serine proteases as therapeutic targets together with the identification, within the wide array of natural BBI variants, of the most potent and specific protease inhibitors, are necessary to better understand the potential of this protein family as colorectal chemopreventive agents.

Soybean Bowman-Birk protease inhibitor (BBI): identification of the mechanisms of BBI suppressive effect on growth of two adenocarcinoma cell lines: AGS and HT29.

BACKGROUND AND AIMS: Bowman-Birk protease inhibitor (BBI) has been well known to suppress the emergence and progression of different cancers. In the present study, the mechanisms by which BBI alters cancers have been addressed. To reach this goal, the effects of BBI on proliferation of and VEGF secretion by two cell lines (AGS: gastric adenocarcinoma and HT-29: colorectal adenocarcinoma) and also BBI effect on MMP-2 and 9 synthesis/secretion by AGS cells was evaluated. METHODS: ELISA method was used to assess VEGF concentration and gelatin zymography was used to address MMP-2 and 9 production/excretion. RESULTS: BBI had powerful inhibitory effect on proliferation and VEGF secretion by both cell lines. In addition, inhibition of MMP-2 and MMP-9 secreted by AGS cells suggests BBI as a potent inhibitor of gastric cancer progression. On the other hand, the results indicated that inhibition of MMP-2, MMP-9 and VEGF secretion is one of the mechanisms of anti-angiogenic effect of BBI. CONCLUSION: BBI expresses powerful suppressive effect on tumor progression of two prevalent cancers: gastric adenocarcinoma and colorectal adenocarcinoma.

Not significant but important.

Armstrong and colleagues report the result of a large Phase IIb randomized trial evaluating the effectiveness of a preparation of the Bowman Birk Inhibitor compared with an oral placebo in reversing the extent of oral leukoplakia as measured visually by pathology or a battery of intermediate end points. In this editorial, we review the report of this negative clinical trials result to highlight the clinical trial process used in evaluating this previously promising chemoprevention agent. Publishing this report is important to address concerns with publication bias. The challenges in running a chemoprevention trial are reviewed with suggestions to enhance progress going forward. Conceptually, developing drugs to intercept the early stages of carcinogenesis is very attractive, but progress in this area has been slow. Two opportunities to overcome this reality are discussed. These measures include the broader use of neoadjuvant, window-of-opportunity trials with new candidate chemoprevention agents to get more textured information about the mechanistic impact of the drug exposure in previously untreated early tumor tissue. In addition, we discuss the use of new intermediate end point markers such as with optical imaging tools to obtain a more objective and quantitative assessment of drug response.

Optimizing biomarkers and endpoints in oral cancer chemoprevention trials.

Chemoprevention, defined as the use of natural, synthetic, or biologic compounds to halt, reverse, or prevent the initial phases of carcinogenesis or the progression of neoplastic cells to cancer, has produced successes, but progress has been slow. Notably, in the field of oral cancer prevention and despite extensive clinical investigations, a standard systemic therapy for patients with oral premalignant lesions is yet to be developed. In view of safety concerns surrounding the use of pharmaceuticals, the use of phytochemicals derived from the diet has been considered but has not yet translated into clinical success. The Bowman Birk Inhibitor (BBI) is a serine protease inhibitor isolated from soybeans possessing domains with trypsin and chymotrypsin inhibitory activity. Encouraging results were previously reported in a phase IIa trial of BBI complex in patients with oral leukoplakia with measurable clinical responses and favorable biomarker changes. In this issue of the journal, the less promising results of the randomized, placebo-controlled phase IIb trial are presented. In this commentary, the complexities involved in defining optimal biomarkers and endpoints for oral cancer prevention trials and the development of dietary chemoprevention agents are discussed.

Bowman birk inhibitor concentrate and oral leukoplakia: a randomized phase IIb trial.

Oral premalignancy serves as an ideal model for study of chemopreventive agents. Although 13-cis-retinoic acid showed reversal of oral premalignancy, toxicity, and reversal of clinical response after cessation of therapy obviated its widespread use. A search for nontoxic agents with cancer preventive activity led us to evaluate Bowman Birk Inhibitor (BBI) formulated as BBI Concentrate (BBIC). We previously reported encouraging results in a phase IIa trial of BBIC in patients with oral leukoplakia with measurable clinical responses and favorable biomarker changes. On the basis of these results, we undertook a randomized, placebo controlled phase IIb trial with patients receiving BBIC or placebo for 6 months, with assessment of clinical response and change in lesion area as primary end point and an intent-to-treat analysis. One hundred and thirty two subjects were randomized; and 89 subjects completed six months on study drug or placebo. Both placebo and BBIC showed a statistically significant decrease in mean lesion area of 17.1% and 20.6%, respectively, and partial or greater clinical responses of 30% and 28% respectively. No significant difference between placebo and study drug arms was observed. Histologic review, review of photographs of lesions, and comparison of serum neu protein and oral mucosal cell protease activity also did not show significant differences between study arms. Probable reasons for these negative results were considered, are discussed, and include a placebo with non-BBIC clinical activity and reduced pharmacokinetic availability of the second batch of BBIC. This experience should be a strong cautionary note to those considering "Green" chemoprevention.

Role of serine proteases in inflammation: Bowman-Birk protease inhibitor (BBI) as a potential therapy for autoimmune diseases.

Serine proteases, a sub-category of the protease family, participate in various physiologic and pathologic conditions. Serine proteases are involved in different arms of the immune system and play an important role in inflammation. They have been evaluated as therapeutic targets in several inflammatory diseases. The Bowman-Birk protease inhibitor (BBI), a soybean-derived serine protease inhibitor, is resistant to temperature and acidic conditions. These characteristics make it a good candidate for oral administration, with no major side effects. In addition, the therapeutic effect of BBI has been shown in inflammatory diseases and cancer. We have demonstrated the immunoregulatory and anti-inflammatory effects of BBI in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Here we review the role of serine proteases in inflammatory diseases, with emphasis on the potential of BBI as a novel oral therapy for multiple sclerosis.

Bowman-Birk inhibitors from legumes and human gastrointestinal health: current status and perspectives.

Bowman-Birk inhibitors and their variants (BBI) from legumes, such as soybean, pea, lentil and chickpea, are a class of naturally-occurring protease inhibitors which have potential health-promoting properties within the gastrointestinal tract. BBI can resist both acidic conditions and the action of proteolytic enzymes, and transit through the stomach and small intestine without major degradation, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. These potential pharmacological benefits have been linked recently to the intrinsic ability of BBI to inhibit serine proteases, and the data suggest that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI. However, the therapeutic targets and the action mechanisms of BBI remain unknown. Their elucidation will provide insights into the properties of these plant protease inhibitors as colorectal chemopreventive agents, providing a strong base for the development of legume crops and their products as pro-nutritional, health-promoting food. The deployment of modern genomic tools and genome sequence information are underpinning studies of natural and induced polymorphism in BBI. Genetic markers for BBI variants with improved properties can be exploited ultimately in legume breeding programmes to assist the introgression of such variant genes and the development of superior genotypes for human nutrition.

Bowman-Birk inhibitor suppresses autoimmune inflammation and neuronal loss in a mouse model of multiple sclerosis.

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor. BBI concentrate (BBIC) is an extract enriched with BBI, but predominantly contains other ingredients including several protease inhibitors. We previously found that BBIC administration to Lewis rats with experimental autoimmune encephalomyelitis (EAE) significantly suppresses disease. In the present study we determined whether BBI mediates the suppressive effects of BBIC in EAE, evaluated its potential neuroprotective effects, and investigated mechanisms of BBI action. We tested effects of purified BBI on clinical and histopathological parameters of EAE in two models (relapsing/remitting EAE in SJL/J mice and chronic EAE in C57BL/6 mice). Effects of BBI were compared to BBIC in relapsing/remitting EAE, and effects of BBI on neuronal survival were examined during acute optic neuritis. Treatment with BBI in both EAE models significantly improved EAE disease parameters (onset, severity, weight loss, inflammation and demyelination). BBI significantly reduced the incidence of optic neuritis and prevented loss of retinal ganglion cells. In most experiments proliferation of immune cells derived from BBI-treated mice was significantly lower relative to control groups. Using Boyden's chamber assay we found that BBI inhibited invasiveness of activated splenocytes through the matrigel barrier. BBI also induced higher production of EAE-suppressive cytokine IL-10 by immune cells. These results demonstrate that BBI is the active component of BBIC that ameliorates clinical EAE. BBI reduces inflammation and attenuates neuronal loss, making it an excellent candidate for oral therapy in MS. BBI likely ameliorates EAE by inhibiting multiple pathways involved in disease pathogenesis.

Bowman-Birk inhibitor concentrate: a novel therapeutic agent for patients with active ulcerative colitis.

Bowman-Birk inhibitor concentrate (BBIC), a soy extract with high protease inhibitor activity, is efficacious in the treatment of colitis in mice and has been used in numerous clinical trials. A randomized, double blind, placebo-controlled trial was performed to investigate the safety and possible benefits of BBIC in patients with active ulcerative colitis. The Sutherland Disease Activity Index (SDAI) was used to assess disease activity, response (Index decrease > or = 3), and remission (Index < or = 1 with no rectal bleeding) in patients receiving 12 weeks of therapy. The Index scores of patients receiving BBIC decreased more than those of the patients receiving placebo (P = 0.067). Beneficial trends were observed in the rates of remission (P = 0.082) and clinical response (P = 0.22). No severe adverse events were observed. This trial suggests a potential benefit over placebo for both achieving clinical response and induction of remission in patients with active ulcerative colitis without apparent toxicity.

Bowman-Birk inhibitor concentrate prevents atrophy, weakness, and oxidative stress in soleus muscle of hindlimb-unloaded mice.

Antigravity muscles atrophy and weaken during prolonged mechanical unloading caused by bed rest or spaceflight. Unloading also induces oxidative stress in muscle, a putative cause of weakness. We tested the hypothesis that dietary supplementation with Bowman-Birk inhibitor concentrate (BBIC), a soy protein extract, would oppose these changes. Adult mice were fed a diet supplemented with 1% BBIC during hindlimb unloading for up to 12 days. Soleus muscles of mice fed the BBIC-supplemented diet weighed less, developed less force per cross-sectional area, and developed less total force after unloading than controls. BBIC supplementation was protective, blunting decrements in soleus muscle weight and force. Cytosolic oxidant activity was assessed using 2',7'-dichlorofluorescin diacetate. Oxidant activity increased in unloaded muscle, peaking at 3 days and remaining elevated through 12 days of unloading. Increases in oxidant activity correlated directly with loss of muscle mass and were abolished by BBIC supplementation. In vitro assays established that BBIC directly buffers reactive oxygen species and also inhibits serine protease activity. We conclude that dietary supplementation with BBIC protects skeletal muscle during prolonged unloading, promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness.

Chronicles in drug discovery.

Chronicles in Drug Discovery features special interest reports on advances in drug discovery and development. This month we focus on the progress of the ongoing search for safe and effective chemopreventive agents. Chemoprevention is a strategy to decrease the risk of developing cancer by using agents that prevent or abrogate carcinogenic processes. Bowman- Birk inhibitor concentrate, budesonide, NCX-4016 and statins are all undergoing investigation in the clinical setting as potential chemopreventive agents for head and neck, lung, colon and breast cancers, respectively.

Biological significance of polymorphism in legume protease inhibitors from the Bowman-Birk family.

Naturally occurring protease inhibitors (PI) of the Bowman-Birk type constitute a major PI family in cereal and legume seeds. The family name is derived from the names of the two investigators who characterised the first inhibitor of this type, the Bowman-Birk inhibitor from soybean (BBI). These proteins have the capacity to inhibit one or more of a range of serine proteases, including the digestive enzymes trypsin and chymotrypsin. PI from this family interact with the active sites of serine proteases in a 'canonical', i.e. substrate-like, manner via exposed reactive site loops of conserved conformation within the inhibitor. Multiple BBI variants can be found within and among species. A limited number of amino acids located within the inhibitory domain is responsible for the primary functional and biological activities of BBI-like proteins. However, sequence variation in binding loops, post-translational modifications at the amino- and carboxy-terminal ends, as well as differences in the multimeric nature of the inhibitors may act in combination to influence the functional properties and the physiological role of BBI-like proteins. Recently, BBI and proteins homologous to BBI (BBI-like proteins) have emerged as highly promising cancer chemopreventive agents. BBI has been shown to be capable of preventing or suppressing carcinogenic processes in a wide variety of in vitro and in vivo animal model systems. The potential exploitation of BBI-like proteins in human health-promotion programmes will depend on elucidating in detail the molecular basis for the variation in biological activities among the many variant forms. New knowledge, derived both from the use of synthetic cyclic peptides that mimic the inhibitory loops of BBI-like proteins, and from genomic data pertaining to the structure of BBI gene classes, together facilitate the manipulation, screening and selection of appropriate variants through biotechnology.

Restoration of connexin 43 by Bowman-Birk protease inhibitor in M5076 bearing mice.

The present study was designed to investigate the effects of Bowman-Birk inhibitor (BBI) on up-regulation of connexin (Cx) expression to estimate BBI's tumor-suppressor effect in mice with M5076 ovarian sarcoma. The relative tumor weight (p<0.05, r(2)=0.301) and proliferating cell nuclear antigen (PCNA, p<0.01, r(2)=0.493) were negatively correlated with the doses of BBI. In contrast, the relative density of Cx43 was positively correlated with the doses of BBI (p<0.05, r(2)=0.351). Therefore, it suggests that the anti-carcinogenic effects of BBI induced negative growth control caused by the expression of Cx43 genes in mice with M5076 ovarian sarcoma.

Selective radioprotection of normal tissues by Bowman-birk proteinase inhibitor (BBI) in mice.

BACKGROUND AND PURPOSE: The efficacy of radiotherapy is limited by the response of normal tissues within the radiation field. The application of normal-tissue-specific radioprotectors may improve the therapeutic benefit of radiotherapy. The purpose of the present study was to explore the in vivo normal-tissue radioprotective potential of Bowman-Birk proteinase inhibitor (BBI), which acts as a normal-cell-specific radioprotector in vitro. MATERIAL AND METHODS: The leg contracture assay in mice, a model system assessing radiation-induced fibrotic processes, was used. To determine whether BBI acts also as a radioprotector of tumors (i. e., FSA and FSAII), the tumor growth delay assay was used. RESULTS: Radiation induced leg contracture in mice with a maximum of about 8 mm at day 150 after irradiation. Treatment of mice with 100 mg/kg BBI before irradiation reduced leg contracture by > 4 mm, by about 50% (p < 0.05, t-test). Doses < 100 mg/kg were ineffective, and doses > 100 mg/kg did not further increase the degree of radioprotection. By contrast, BBI did not induce radioprotection of either TP53-mutated FSA or TP53-normal FSAII tumor xenografts in mice, which argues for normal-tissue-specific effect. CONCLUSION: BBI acts as a potent selective normal-tissue radioprotector in vitro and in vivo, apparently without protecting tumors, and thus has the potential to improve clinical radiotherapy.