Snake and arthropod venoms: Search for inflammatory activity in human cells involved in joint diseases.

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

A Kunitz-type inhibitor from tick salivary glands: A promising novel antitumor drug candidate.

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Losac and Lopap Recombinant Proteins from Lonomia obliqua Bristles Positively Modulate the Myoblast Proliferation Process.

The pursuit of better therapies for disorders creating deficiencies in skeletal muscle regeneration is in progress, and several biotoxins are used in skeletal muscle research. Since recombinant proteins derived from Lonomia obliqua bristles, recombinant Lonomia obliqua Stuart-factor activator (rLosac) and recombinant Lonomia obliqua prothrombin activator protease (rLopap) act as cytoprotective agents and promote cell survival, we hypothesize that both rLosac and rLopap favour the skeletal muscle regeneration process. In the present work, we investigate the ability of these recombinant proteins rLosac and rLopap to modulate the production of key mediators of the myogenic process. The expression of myogenic regulatory factors (MRFs), cell proliferation, the production of prostaglandin E2 (PGE2) and the protein expression of cyclooxygenases COX-1 and COX-2 were evaluated in C2C12 mouse myoblasts pre-treated with rLosac and rLopap. We found an increased proliferation of myoblasts, stimulated by both recombinant proteins. Moreover, these proteins modulated PGE2 release and MRFs activities. We also found an increased expression of the EP4 receptor in the proliferative phase of C2C12 cells, suggesting the involvement of this receptor in the effects of PGE2 in these cells. Moreover, the recombinant proteins inhibited the release of IL-6 and PGE2, which is induced by an inflammatory stimulus by IL-1ß. This work reveals rLopap and rLosac as promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury.

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.

The phytoactive constituents of Eugenia selloi B.D. Jacks (pitangatuba): Toxicity and elucidation of their anti-inflammatory mechanism(s) of action.

We determined the phytochemical composition, anti-inflammatory mechanism of action, ROS/RNS scavenging capacity and systemic toxicity of a purified subfraction (S8) of Eugenia selloi. The composition of S8 was assessed by LC-ESI-QTOF-MS; the anti-inflammatory activity in RAW264.7 macrophages through NF-κB activation and biomarkers by multiplex in THP-1 cells; neutrophil migration, intravital microscopy and ICAM-1 expression in mice; NETs formation and CD11b expression; S8 scavenging capacity of ROS/RNS; toxicity in Galleria mellonella larvae model. Coumaric acid, quercetrin and vanillic acid were identified. S8 decreased NF-κB activation, IL-1ß, IL-6, IL-10, MDC and MCP-1 levels, reduced neutrophil migration and ICAM-1 expression in mice; S8 did not interfere NET formation and CD11b expression, exhibited high antioxidant and showed negligible toxicity. E. selloi proved to be a promising, yet underexplored source of bioactive compounds, which can be useful employed in agribusiness and in the pharmaceutical and food industry to develop new products or human health supplies.

Lonomia obliqua Envenoming and Innovative Research.

As a tribute to Butantan Institute in its 120th anniversary, this review describes some of the scientific research efforts carried out in the study of Lonomia envenoming in Brazil, a country where accidents with caterpillars reach over 42,000 individuals per year (especially in South and Southeast Brazil). Thus, the promising data regarding the studies with Lonomia's toxins contributed to the creation of new research centers specialized in toxinology based at Butantan Institute, as well as to the production of the antilonomic serum (ALS), actions which are in line with the Butantan Institute mission "to research, develop, manufacture, and provide products and services for the health of the population". In addition, the study of the components of the Lonomia obliqua bristle extract led to the discovery of new molecules with peculiar properties, opening a field of knowledge that could lead to the development and innovation of new drugs aimed at cell regeneration and inflammatory diseases.

Lonomia obliqua Venom Induces NF-κB Activation and a Pro-Inflammatory Profile in THP-1-Derived Macrophage.

Envenomation caused by contact with Lonomia obliqua bristles is characterized by pain, an intense systemic proinflammatory reaction and disturbances in the coagulation cascade that can cause severe clinical manifestations and death. However, the role of immune system components in these effects is still poorly understood. In this study, we evaluated the cytotoxic effect of L. obliqua venom on THP-1-derived macrophages and its ability to modulate inflammatory markers, as well as the cytokine and chemokine release profile. Our results show that L. obliqua venom is able to directly exert a potent pro-inflammatory reaction in macrophages, characterized by the activation of the NF-κB transcription factor pathway, the expression of CD80 and CD83, and the release of pro-inflammatory mediators such as TNF-α, IL-1ß, IL-6, IL-8 and CXCL10. These results suggest that macrophages can play an important role during the orchestration of the inflammatory response present in envenomation caused by Lonomia obliqua caterpillars.

Neuroprotective effect of rLosac on supplement-deprived mouse cultured cortical neurons involves maintenance of monocarboxylate transporter MCT2 protein levels

The recombinant Lonomia obliqua Stuart-factor activator (rLosac) is a recombinant hemolin which belongs to the immunoglobulin superfamily of cell adhesion molecules. It is capable of inducing pro-survival activity in serum-deprived human umbilical vein endothelial cells (HUVECs) and fibroblasts by increasing mitochondrial metabolism. We hypothesize that it could promote neuronal survival by acting on neuroenergetics. Our study reveals that treatment of primary mouse cortical neurons cultured in neurobasal medium lacking B27 supplement with rLosac led to an enhancement of cell viability in a time- and concentration-dependent manner. In parallel, preserved or enhanced phosphorylation of Akt, p44, and p42 MAPK, as well as mTOR was observed following treatment with rLosac. During deprivation, as assessed by western blot and qRT-PCR, protein and mRNA expression of MCT2 (the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate) decreased significantly in B27 supplement-deprived cortical neurons and was hardly detected after 24h of deprivation. Interestingly, rLosac maintained MCT2 protein expression after 24h of deprivation including at the cell surface without preventing mRNA loss. MCT2 knockdown reduced rLosac-enhanced cell viability, confirming its involvement in rLosac effect. Enhanced uptake of lactate was detected following rLosac treatment and might contribute to rLosac-enhanced viability during deprivation. In the presence of both lactate and rLosac, cell viability was higher than in the presence of lactate alone. Our observations suggest that rLosac promotes cell viability in stressed (B27 supplement-deprived) neurons by facilitating the use of lactate as energy substrate via the preservation of MCT2 protein expression.

Neuroprotective effect of rLosac on supplement-deprived mouse cultured cortical neurons involves maintenance of monocarboxylate transporter MCT2 protein levels

The recombinant Lonomia obliqua Stuart-factor activator (rLosac) is a recombinant hemolin which belongs to the immunoglobulin superfamily of cell adhesion molecules. It is capable of inducing pro-survival activity in serum-deprived human umbilical vein endothelial cells (HUVECs) and fibroblasts by increasing mitochondrial metabolism. We hypothesize that it could promote neuronal survival by acting on neuroenergetics. Our study reveals that treatment of primary mouse cortical neurons cultured in neurobasal medium lacking B27 supplement with rLosac led to an enhancement of cell viability in a time- and concentration-dependent manner. In parallel, preserved or enhanced phosphorylation of Akt, p44, and p42 MAPK, as well as mTOR was observed following treatment with rLosac. During deprivation, as assessed by western blot and qRT-PCR, protein and mRNA expression of MCT2 (the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate) decreased significantly in B27 supplement-deprived cortical neurons and was hardly detected after 24h of deprivation. Interestingly, rLosac maintained MCT2 protein expression after 24h of deprivation including at the cell surface without preventing mRNA loss. MCT2 knockdown reduced rLosac-enhanced cell viability, confirming its involvement in rLosac effect. Enhanced uptake of lactate was detected following rLosac treatment and might contribute to rLosac-enhanced viability during deprivation. In the presence of both lactate and rLosac, cell viability was higher than in the presence of lactate alone. Our observations suggest that rLosac promotes cell viability in stressed (B27 supplement-deprived) neurons by facilitating the use of lactate as energy substrate via the preservation of MCT2 protein expression.

Exploring the in vivo wound healing effects of a recombinant hemolin from the caterpillar Lonomia obliqua.

BACKGROUND: Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. METHODS: Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. RESULTS: The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. CONCLUSION: The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.

Hemolin triggers cell survival on fibroblasts in response to serum deprivation by inhibition of apoptosis.

Fibroblasts are the main cellular component of connective tissues and play important roles in health and disease through the production of collagen, fibronectin and growth factors. Under certain conditions, such as wound healing, fibroblasts intensify their metabolic demand, while the restriction of nutrients affect matrix composition, cell metabolism and behavior. In lepidopterans, wound healing is regulated by ecdysteroid hormones, which upregulate multifunctional proteins such as hemolin. However, the role of hemolin in cell proliferation and wound healing is not clear. rLosac is a recombinant hemolin from the caterpillar Lonomia obliqua whose proliferative and cytoprotective effects on endothelial cells have been described. Here, we show that rLosac induces a marked cell survival effect on fibroblast submitted to serum deprivation, which is observable as early as 24h, as demonstrated through the MTT assay, as well as an increase in migration of human dermal fibroblasts (HDF). No effects on cell proliferation or cell cycle distribution of fibroblasts in normal conditions were observed, suggesting that rLosac induces an effect in stressful conditions such serum deprivation but not when nutrient are sufficient. By flow cytometry, rLosac caused an apparent dose-dependent increase in cells in the S phase of the cell cycle and a significant reduction of cells with fragmented DNA. Furthermore, treatment with rLosac results in a significant decrease in the production of reactive oxygen species and in the loss of mitochondrial membrane potential, indicating that a reduction in oxidative stress is involved in rLosac-mediated cytoprotection. Our results also show an up-regulation of Bcl-2 and a down-regulation of Bax protein levels, inhibition of cytochrome c release and a reduction in caspase-3 levels, all considered critical factors for apoptosis. Moreover, rLosac treatment reduces the morphological changes induced by prolonged serum deprivation including the emergence of apoptotic bodies, nucleus fragmentation, cytoplasmic vacuolization and loss of extracellular matrix organization. The wound scratch test assay revealed that rLosac could enhance wound healing in vitro. Altogether, these findings suggest that rLosac strongly induces cellular protection in conditions of stress by serum deprivation preventing damage and loss of mitochondrial function by inhibiting apoptosis. This finding opens a new perspective to further understand the role of hemolin proteins during cellular processes such as wound healing and development.

Exploring the in vivo wound healing effects of a recombinant hemolin from the caterpillar Lonomia obliqua

Background Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. Methods Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.(AU)

Exploring the in vivo wound healing effects of a recombinant hemolin from the caterpillar Lonomia obliqua

Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolins roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosacs in vivo effects using a skin wound healing model in rats. Methods Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.

Exploring the in vivo wound healing effects of a recombinant hemolin from the caterpillar Lonomia obliqua

Background: Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. Methods: Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results: The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion: The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations

Hemolin triggers cell survival on fibroblasts in response to serum deprivation by inhibition of apoptosis

Fibroblasts are the main cellular component of connective tissues and play important roles in health and disease through the production of collagen, fibronectin and growth factors. Under certain conditions, such as wound healing, fibroblasts intensify their metabolic demand, while the restriction of nutrients affect matrix composition, cell metabolism and behavior. In lepidopterans, wound healing is regulated by ecdysteroid hormones, which upregulate multifunctional proteins such as hemolin. However, the role of hemolin in cell proliferation and wound healing is not clear. rLosac is a recombinant hemolin from the caterpillar Lonomia obliqua whose proliferative and cytoprotective effects on endothelial cells have been described. Here, we show that rLosac induces a marked cell survival effect on fibroblast submitted to serum deprivation, which is observable as early as 24 h, as demonstrated through the MTT assay, as well as an increase in migration of human dermal fibroblasts (HDF). No effects on cell proliferation or cell cycle distribution of fibroblasts in normal conditions were observed, suggesting that rLosac induces an effect in stressful conditions such serum deprivation but not when nutrient are sufficient. By flow cytometry, rLosac caused an apparent dose-dependent increase in cells in the S phase of the cell cycle and a significant reduction of cells with fragmented DNA. Furthermore, treatment with rLosac results in a significant decrease in the production of reactive oxygen species and in the loss of mitochondrial membrane potential, indicating that a reduction in oxidative stress is involved in rLosac-mediated cytoprotection. Our results also show an up-regulation of Bcl-2 and a down-regulation of Bax protein levels, inhibition of cytochrome c release and a reduction in caspase-3 levels, all considered critical factors for apoptosis. Moreover, rLosac treatment reduces the morphological changes induced by prolonged serum deprivation including the emergence of apoptotic bodies, nucleus fragmentation, cytoplasmic vacuolization and loss of extracellular matrix organization. The wound scratch test assay revealed that rLosac could enhance wound healing in vitro. Altogether, these findings suggest that rLosac strongly induces cellular protection in conditions of stress by serum deprivation preventing damage and loss of mitochondrial function by inhibiting apoptosis. This finding opens a new perspective to further understand the role of hemolin proteins during cellular processes such as wound healing and development. (C) 2016 Elsevier Masson SAS. All rights reserved.

Amblyomin-X having a Kunitz-type homologous domain, is a noncompetitive inhibitor of FXa and induces anticoagulation in vitro and in vivo

Background: Cancer has long been associated with thrombosis and many of the standard chemotherapeutics used to treat cancer are pro-thrombotic. Thus, the identification of novel selective anticancer drugs that also have antithrombotic properties is of enormous significance. Amblyomin-X is an anticancer protein derived from the salivary glands of the Amblyomma cajennense tick. Methods: In this work, we determined the inhibition profile of Amblyomin-X and its effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), using various approaches such as, kinetic analyses, amidolytic assays, SDS-PAGE, and mass spectrometry. Results: Amblyomin-X inhibited factor Xa, prothrombinase and tenase activities. It was hydrolyzed by trypsin and plasmin. MS/MS data of tryptic hydrolysate of Amblyomin-X suggested the presence of Cys(8)-Cys(59) and Cys(19)-Cys(42) but not Cys(34)-Cys(55) disulfide bond. Instead of Cys(34)-Cys(55), two noncanonical Cys(34)-Cys(74) and Cys(55)-Cys(74) disulfide bonds were identified. Furthermore, when Amblyomin-X (1 mg/kg) injected in rabbits, it prolonged aPTT and PT. Conclusion: Amblyomin-X is a noncompetitive inhibitor (K-i = 3.9 mu M) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The disulfide Cys(34)-Cys(55) bond probably scrambles with inter chain seventh free cysteine residues (Cys(74)) of Amblyomin-X. The prolongation of PT and aPTT is reversible. General Significance. In term of anticoagulant property, this is structural and functional characterization of Amblyomin-X. All together, these results and previous findings suggest that Amblyomin-X has a potential to become an anticancer drug with antithrombotic property. (C) 2016 Elsevier B.V. All rights reserved.