Unveiling the bioactivity and bioaccessibility of phenolic compounds from organic coffee husks using an in vitro digestion model.

BACKGROUND: The large quantities of by-products generated in the coffee industry are a problem. Studies related to the biological potential of organic coffee husks are still limited. The aim of this work was to investigate the occurrence of phenolic compounds in organic coffee husks and to evaluate their potential as a source of bioactive dietary components. RESULTS: To achieve this objective, three extracts were prepared, namely extractable polyphenols (EPs), hydrolyzable non-extractable polyphenols (H-NEPs), and non-extractable polyphenols (NEPs). These extracts were characterized and evaluated for their bioactive properties after simulated gastrointestinal digestion. The results show that the extraction process affected the occurrence of phenols from coffee peels, especially for caffeic acid, gallic acid, and chlorogenic acid. The free and bound polyphenols found in the extracts and digests not only showed antioxidant properties against 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals but were also strongly bioavailable and had good anticoagulant potential. CONCLUSION: These results highlight the potential health benefits of phytochemicals from coffee husks and open new perspectives for the use of such compounds in dietary supplements. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time.

BACKGROUND: (p-BthTX-I)2 K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required. OBJECTIVES: To determine whether (p-BthTX-I)2 K affects the hemostatic system. METHODS: Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I)2 K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg- 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I)2 K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I)2 K (4.0 mg/kg and 8.0 mg/kg) or saline administration. RESULTS: (p-BthTX-I)2 K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I)2 K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I)2 K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I)2 K did not prolong the bleeding time in the mouse model of arterial thrombosis. CONCLUSION: These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I)2 K possibly by kallikrein inhibition, suggesting its strong biotechnological potential.

Impairment of SK-MEL-28 Development-A Human Melanoma Cell Line-By the Crataeva tapia Bark Lectin and Its Sequence-Derived Peptides.

Melanoma is difficult to treat with chemotherapy, prompting the need for new treatments. Protease inhibitors have emerged as promising candidates as tumor cell proteases promote metastasis. Researchers have developed a chimeric form of the Bauhinia bauhinioides kallikrein inhibitor, rBbKIm, which has shown negative effects on prostate tumor cell lines DU145 and PC3. Crataeva tapia bark lectin, CrataBL, targets sulfated oligosaccharides in glycosylated proteins and has also demonstrated deleterious effects on prostate and glioblastoma tumor cells. However, neither rBbKIm nor its derived peptides affected the viability of SK-MEL-28, a melanoma cell line, while CrataBL decreased viability by over 60%. Two peptides, Pep. 26 (Ac-Q-N-S-S-L-K-V-V-P-L-NH2) and Pep. 27 (Ac-L-P-V-V-K-L-S-S-N-Q-NH2), were also tested. Pep. 27 suppressed cell migration and induced apoptosis when combined with vemurafenib, while Pep. 26 inhibited cell migration and reduced nitric oxide and the number of viable cells. Vemurafenib, a chemotherapy drug used to treat melanoma, was found to decrease the release of interleukin 8 and PDGF-AB/BB cytokines and potentiated the effects of proteins and peptides in reducing these cytokines. These findings suggest that protease inhibitors may be effective in blocking melanoma cells and highlight the potential of CrataBL and its derived peptides.

Effects of a Peptide Derived from the Primary Sequence of a Kallikrein Inhibitor Isolated from Bauhinia bauhinioides (pep-BbKI) in an Asthma-COPD Overlap (ACO) Model.

(1) There are several patients with asthma-COPD overlap (ACO). A peptide derived from the primary sequence of a kallikrein inhibitor isolated from Bauhinia bauhinioides (pep-BbKI) has potent anti-inflammatory and antioxidant effects. Purpose: To investigate the effects of pep-BbKI treatment in an ACO model and compare them with those of corticosteroids. (2) BALB/c mice were divided into groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep-BbKI (treated with inhibitor), ACO-DX (dexamethasone treatment), ACO-DX-pep-BbKI (both treatments), and SAL-pep-BbKI (saline group treated with inhibitor). We evaluated: hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), exhaled nitric oxide (eNO), IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, MMP-9, MMP-12, TGF-ß, collagen fibers, iNOS, eNO, linear mean intercept (Lm), and NF-κB in airways (AW) and alveolar septa (AS). (3) ACO-pep-BbKI reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, neutrophils, IL-5, IL-10, IL-17, IFN-γ, TNF-α, MMP-12 (AW), collagen fibers, iNOS (AW), and eNO (p > 0.05). ACO-DX reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, total cells and differentials, IL-1ß(AS), IL-5 (AS), IL-6 (AS), IL-10 (AS), IL-13 (AS), IFN-γ, MMP-12 (AS), TGF-ß (AS), collagen fibers (AW), iNOS, and eNO (p > 0.05). SAL was similar to SAL-pep-BbKI for all comparisons (p > 0.05). (4) Pep-BbKI was similar to dexamethasone in reducing the majority of alterations of this ACO model.

Investigating the Effects of a New Peptide, Derived from the Enterolobium contortisiliquum Proteinase Inhibitor (EcTI), on Inflammation, Remodeling, and Oxidative Stress in an Experimental Mouse Model of Asthma-Chronic Obstructive Pulmonary Disease Overlap (ACO).

The synthesized peptide derived from Enterolobium contortisiliquum (pep3-EcTI) has been associated with potent anti-inflammatory and antioxidant effects, and it may be a potential new treatment for asthma-COPD overlap-ACO). Purpose: To investigate the primary sequence effects of pep3-EcTI in an experimental ACO. BALB/c mice were divided into eight groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep3-EcTI (treated with inhibitor), ACO-DX (treated with dexamethasone), ACO-DX-pep3-EcTI (treated with dexamethasone and inhibitor), and SAL-pep3-EcTI (saline group treated with inhibitor). We evaluated the hyperresponsiveness to methacholine, exhaled nitric oxide, bronchoalveolar lavage fluid (BALF), mean linear intercept (Lm), inflammatory markers, tumor necrosis factor (TNF-α), interferon (IFN)), matrix metalloproteinases (MMPs), growth factor (TGF-ß), collagen fibers, the oxidative stress marker inducible nitric oxide synthase (iNOS), transcription factors, and the signaling pathway NF-κB in the airways (AW) and alveolar septa (AS). Statistical analysis was conducted using one-way ANOVA and t-tests, significant when p < 0.05. ACO caused alterations in the airways and alveolar septa. Compared with SAL, ACO-pep3-EcTI reversed the changes in the percentage of resistance of the respiratory system (%Rrs), the elastance of the respiratory system (%Ers), tissue resistance (%Gtis), tissue elastance (%Htis), airway resistance (%Raw), Lm, exhaled nitric oxide (ENO), lymphocytes, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, transforming growth factor (TGF)-ß, collagen fibers, and iNOS. ACO-DX reversed the changes in %Rrs, %Ers, %Gtis, %Htis, %Raw, total cells, eosinophils, neutrophils, lymphocytes, macrophages, IL-1ß, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, TGF-ß, collagen fibers, and iNOS. ACO-DX-pep3-EcTI reversed the changes, as was also observed for the pep3-EcTI and the ACO-DX-pep3-EcTI. Significance: The pep3-EcTI was revealed to be a promising strategy for the treatment of ACO, asthma, and COPD.

Plant Kunitz Inhibitors and Their Interaction with Proteases: Current and Potential Pharmacological Targets.

The action of proteases can be controlled by several mechanisms, including regulation through gene expression; post-translational modifications, such as glycosylation; zymogen activation; targeting specific compartments, such as lysosomes and mitochondria; and blocking proteolysis using endogenous inhibitors. Protease inhibitors are important molecules to be explored for the control of proteolytic processes in organisms because of their ability to act on several proteases. In this context, plants synthesize numerous proteins that contribute to protection against attacks by microorganisms (fungi and bacteria) and/or invertebrates (insects and nematodes) through the inhibition of proteases in these organisms. These proteins are widely distributed in the plant kingdom, and are present in higher concentrations in legume seeds (compared to other organs and other botanical families), motivating studies on their inhibitory effects in various organisms, including humans. In most cases, the biological roles of these proteins have been assigned based mostly on their in vitro action, as is the case with enzyme inhibitors. This review highlights the structural evolution, function, and wide variety of effects of plant Kunitz protease inhibitors, and their potential for pharmaceutical application based on their interactions with different proteases.

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins.

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

Exosites expedite blood coagulation.

A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a "dock and lock." This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future.

Bauhinia Protease Inhibitors Attenuate Gastric Ulcer by Blocking Neutrophil Enzymes.

Proteases play a pivotal role in many signaling pathways; inhibitors of well-established proteases have shown a substantial therapeutic success. This study aimed to examine the in vivo effects of 3 protease inhibitors isolated from Bauhinia species: i) Bauhinia mollis elastase inhibitor, which blocks human neutrophil elastase (Kiapp 2.8 nM) and cathepsin G (Kiapp 1.0 nM) activities; ii) Bauhinia mollis trypsin inhibitor, a trypsin inhibitor (Kiapp 5.0 nM); and iii) Bauhinia bauhinioides cruzipain inhibitor, which inhibits elastase (Kiapp 2.6 nM), cathepsin G (Kiapp 160.0 nM), and the cysteine proteases cathepsin L (Kiapp 0.2 nM). Bauhinia bauhinioides cruzipain inhibitor, Bauhinia mollis elastase inhibitor, and Bauhinia mollis trypsin inhibitor were isolated using acetone and ammonium sulfate fractionations, DEAE-Sephadex, trypsin-Sepharose, and Resource-Q chromatographies. Mice and rats were treated intraperitoneally with 1 dose of inhibitor; gastric mucosal lesions were induced by cold-restraint stress. Oral pretreatment of mice with Bauhinia mollis elastase inhibitor or Bauhinia mollis trypsin inhibitor (1 - 10 mg/kg) did not show anti-ulcer effect, while Bauhinia bauhinioides cruzipain inhibitor (0.1 - 1.0 mg/kg) produced a similar reduction of the index of mucosal damage at all effective doses (30 to 33% < control). In rats at doses lower than those used in mice, Bauhinia mollis elastase inhibitor and Bauhinia bauhinioides cruzipain inhibitor reduced the index of mucosal damage by 66% and 54% of controls, respectively. The results indicate a protective effect against gastric mucosal lesions associated with elastase inhibition but not inhibition of trypsin activities. Moreover, the lack of Bauhinia mollis elastase inhibitor efficacy observed in mice may possibly be related to the reported structural differences of elastase in mice and rats.

In vitro evaluation of mercury (Hg2+) effects on biofilm formation by clinical and environmental isolates of Klebsiella pneumoniae.

The increase in urbanization and industrialization has contributed to the contamination of different environments by means of xenobiotic compounds, such as heavy metals, causing changes in microbial communities. Among these metals, the Mercury (Hg2+) is one the most prevalent toxic metals for the environment The present study aimed to evaluate the effect of mercury on the formation of biofilm by environmental (collected from urban stream water) and clinical isolates of Klebsiella pneumoniae. In addition, antibiotic resistance, virulence factors, and genetic diversity were investigated. Taxonomic identity of eight isolates (one reference, two clinical, and five environmental isolates) was performed by MALDI-TOF-MS, while the antibiotic susceptibility profile was assessed by the disc diffusion method. The ability to form biofilms was evaluated by culture on Congo red agar and by crystal violet staining. Biofilm structure was analyzed by scanning electron microscopy. The hydrophobicity profile and the presence of the virulence genes cps, fimH, and mrkD was investigated. The presence of merA and its relationship with antimicrobial resistance were also assessed. The identity of all isolates was confirmed by MALDI-TOF-MS, and different profiles of resistance to mercury and antibiotics as well as of biofilm formation were identified for the clinical and environmental isolates. All isolates were hydrophilic and positive for the virulence genes cps, fimH, and mrkD; only the clinical isolate K36-A2 was positive for merA. The diversity of the isolates was confirmed by ERIC-PCR, which revealed high heterogeneity among the isolates. In conclusion, the data demonstrate that the investigated isolates present different responses to exposure to Hg2+ and correspond to distinct populations of K. pneumoniae disseminated in the investigated environment. The data obtained in this work will aid in understanding the mechanisms of survival of this pathogen under adverse conditions.

A Bifunctional Molecule with Lectin and Protease Inhibitor Activities Isolated from Crataeva tapia Bark Significantly Affects Cocultures of Mesenchymal Stem Cells and Glioblastoma Cells.

Currently available drugs for treatment of glioblastoma, the most aggressive brain tumor, remain inefficient, thus a plethora of natural compounds have already been shown to have antimalignant effects. However, these have not been tested for their impact on tumor cells in their microenvironment-simulated cell models, e.g., mesenchymal stem cells in coculture with glioblastoma cell U87 (GB). Mesenchymal stem cells (MSC) chemotactically infiltrate the glioblastoma microenvironment. Our previous studies have shown that bone-marrow derived MSCs impair U87 growth and invasion via paracrine and cell-cell contact-mediated cross-talk. Here, we report on a plant-derived protein, obtained from Crataeva tapia tree Bark Lectin (CrataBL), having protease inhibitory/lectin activities, and demonstrate its effects on glioblastoma cells U87 alone and their cocultures with MSCs. CrataBL inhibited U87 cell invasion and adhesion. Using a simplified model of the stromal microenvironment, i.e., GB/MSC direct cocultures, we demonstrated that CrataBL, when added in increased concentrations, caused cell cycle arrest and decreased cocultured cells' viability and proliferation, but not invasion. The cocultured cells' phenotypes were affected by CrataBL via a variety of secreted immunomodulatory cytokines, i.e., G-CSF, GM-CSF, IL-6, IL-8, and VEGF. We hypothesize that CrataBL plays a role by boosting the modulatory effects of MSCs on these glioblastoma cell lines and thus the effects of this and other natural lectins and/or inhibitors would certainly be different in the tumor microenvironment compared to tumor cells alone. We have provided clear evidence that it makes much more sense testing these potential therapeutic adjuvants in cocultures, mimicking heterogeneous tumor-stroma interactions with cancer cells in vivo. As such, CrataBL is suggested as a new candidate to approach adjuvant treatment of this deadly tumor.

A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice.

Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management.

The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI) Attenuates Elastase-Induced Emphysema in Mice.

Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD). However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI) to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group) or saline (SAL group) and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups). At day 28, the following analyses were performed: (I) lung mechanics, (II) exhaled nitric oxide (ENO), (III) bronchoalveolar lavage fluid (BALF), and (IV) lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment.

Conformational and biological properties of Bauhinia bauhinioides kallikrein inhibitor fragments with bradykinin-like activities.

Proteinase inhibitors extracted form medicinal plants are an interesting source of new drugs. Modifications in the structure of some of this kind of macromolecules could also lead to compounds of interesting biological properties. In this work, we synthesized and tested one fragment containing the reactive site of the Bauhinia bauhinioides kallikrein inhibitor (BbKI), denoted BbKI51-62 , and a related analog (P2 ) in which a proline residue was inserted in order to mimic the N-terminal region of the bradykinin molecule. The related retro-inverso counterparts Ri-BbKI51-62 and Ri-P2 were also included. The ability of these peptides to induce contraction of stomach fundus isolated from mouse was evaluated as well as their capability to induce calcium release from a cell culture of smooth muscle from guinea pig ileum. The conformational properties of the peptides were evaluated by circular dichroism and their resistance to enzymatic degradation by exposure to human blood plasma. Our results show that neither the parent BbKI51-62 nor its Ri-BbKI51-62 analog exhibit bradykinin-like activity, although the retro-inverso isomer was resistant to blood plasma degradation. On the other hand, the peptides P2 and Ri-P2 presented contractile activities on gastric smooth muscle from stomach fundus possibly by acting via B-2 receptor. Both compounds also induce calcium release from guinea pig ileum muscle cells in a manner similar to bradykinin. Moreover, both compounds also inhibited porcine pancreatic kallikrein. However, conformational analysis did not reveal any direct correlation between structure and biological effects.

High serum levels of proinflammatory markers during epileptogenesis. Can omega-3 fatty acid administration reduce this process?

During the epileptogenic process, several events may occur, such as an important activation of the immune system in the central nervous system. The response to seizure activity results in an inflammation in the brain as well as in the periphery. Moreover, CRP and cytokines may be able to interact with numerous ligands in response to cardiac injury caused by sympathetic stimulation in ictal and postictal states. Based on this, we measured the serum levels of C-reactive protein (CRP) and cytokines during acute, silent, and chronic phases of rats submitted to the pilocarpine model of epilepsy. We have also analyzed the effect of a chronic treatment of these rats with omega-3 fatty acid in CRP and cytokine levels, during an epileptic focus generation. C-reactive protein and cytokines such as IL-1ß, IL-6, and TNF-α presented high concentration in the blood of rats, even well after the occurrence of SE. We found reduced levels of CRP and all proinflammatory cytokines in the blood of animals with chronic seizures, treated with omega-3, when compared with those treated with vehicle solution. Taken together, our results strongly suggest that the omega-3 is an effective treatment to prevent SUDEP occurrence due to its capability to act as an anti-inflammatory compound, reducing the systemic inflammatory parameters altered by seizures.

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.

CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation.

Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (Kiapp=8.6 µm). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking coagulation and arterial thrombus formation, without increasing bleeding time.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Effects of plant protease inhibitors (Pep-3-EcTI, Pep-BbKI, and Pep-BrTI) versus corticosteroids on inflammation, remodeling, and oxidative stress in an asthma-COPD (ACO) model.

The peptide derived from E. contortisiliquum trypsin inhibitor (Pep-3-EcTI), peptide derived from kallikrein inhibitor isolated from B. bauhinioides (Pep-BbKI), and B. rufa peptide modified from B. bauhinioides (Pep-BrTI) peptides exhibit anti-inflammatory and antioxidant activities, suggesting their potential for treating asthma-chronic obstructive pulmonary disease (COPD) overlap (ACO). We compared the effects of these peptides with dexamethasone (DX) treatment in an ACO model. In this study, 11 groups of male BALB/c mice were pre-treated under different conditions, including sensitization with intraperitoneal injection and inhalation of ovalbumin (OVA), intratracheal instillation of porcine pancreatic elastase (ELA), sensitization with intraperitoneal injection, and various combinations of peptide treatments with Pep-3-EcTI, Pep-BbKI, Pep-BrTI, dexamethasone, and non-treated controls (SAL-saline). Respiratory system resistance, airway resistance, lung tissue resistance, exhaled nitric oxide, linear mean intercept, immune cell counts in the bronchoalveolar lavage fluid, cytokine expression, extracellular matrix remodeling, and oxidative stress in the airways and alveolar septa were evaluated on day 28. Results showed increased respiratory parameters, inflammatory markers, and tissue remodeling in the ACO group compared to controls. Treatment with the peptides or DX attenuated or reversed these responses, with the peptides showing effectiveness in controlling hyperresponsiveness, inflammation, remodeling, and oxidative stress markers. These peptides demonstrated an efficacy comparable to that of corticosteroids in the ACO model. However, this study highlights the need for further research to assess their safety, mechanisms of action, and potential translation to clinical studies before considering these peptides for human use.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.