A Complex Pattern of Gene Expression in Tissue Affected by Viperid Snake Envenoming: The Emerging Role of Autophagy-Related Genes.

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A2 (PLA2s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor ß (TGF-ß) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings.

Snake Venom Components as Therapeutic Drugs in Ischemic Heart Disease.

Ischemic heart disease (IHD), especially myocardial infarction (MI), is a leading cause of death worldwide. Although coronary reperfusion is the most straightforward treatment for limiting the MI size, it has nevertheless been shown to exacerbate ischemic myocardial injury. Therefore, identifying and developing therapeutic strategies to treat IHD is a major medical challenge. Snake venoms contain biologically active proteins and peptides that are of major interest for pharmacological applications in the cardiovascular system (CVS). This has led to their use for the development and design of new drugs, such as the first-in-class angiotensin-converting enzyme inhibitor captopril, developed from a peptide present in Bothrops jararaca snake venom. This review discusses the potential usefulness of snake venom toxins for developing effective treatments against IHD and related diseases such as hypertension and atherosclerosis. It describes their biological effects at the molecular scale, their mechanisms of action according to their different pharmacological properties, as well as their subsequent molecular pathways and therapeutic targets. The molecules reported here have either been approved for human medical use and are currently available on the drug market or are still in the clinical or preclinical developmental stages. The information summarized here may be useful in providing insights into the development of future snake venom-derived drugs.

Crotoxin Modulates Macrophage Phenotypic Reprogramming.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 µg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 µg/animal: M1 and at 5.0 µg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 µg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors - Benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy.

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Bj-PRO-10c, as an allosteric regulator of argininosuccinate synthase, is a potential therapy for neuroblastoma metastasis.

Cancer is a global public health issue. Neuroblastoma (NB) originates from any tissue of the sympathetic nervous system, and the most affected site is the abdomen. The adrenal gland is the primary site in 38% of cases. Approximately 50% of patients have metastatic disease at diagnosis, and bone marrow is often affected. Metastatic disease is characterized by the spreading of cancer cells that are frequently resistant to chemotherapy and radiotherapy from the primary tumor to other specific parts of the body and is responsible for 90% of cancer-related deaths. Increasing evidence has indicated that nitric oxide (NO) signaling is implicated in the pathophysiology of many types of cancer, particularly in tumorigenesis and cancer progression. However, the effect of NO on metastasis cannot be easily classified as prometastatic or antimetastatic. An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. Here, the proline-rich decapeptide isolated from Bothrops jararaca venom (Bj-PRO-10c) that enhances and sustains the generation of NO was used to unravel the role of metabolic NO in steps of metastasis. Bj-PRO-10c showed an antimetastatic effect, mainly by interfering with actin cytoskeleton rearrangement, controlling cell proliferation, and decreasing the seeding efficiency of NB in metastatic niches. Therefore, we proposed that an approach for controlled NO induction with the right molecular strategies can hopefully inhibit metastasis and increase the lifespan of NB patients.

Oxidative stress induced by Pollonein-LAAO, a new L-amino acid oxidase from Bothrops moojeni venom, prompts prostate tumor spheroid cell death and impairs the cellular invasion process in vitro.

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.

rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis.

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbß3, a fundamental platelet glycoprotein, and αvß3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of ß-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbß3 antagonist, capable of preventing arterial thrombosis.

Effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with dengue virus.

Dengue fever is considered a major public health problem in tropical and subtropical regions. Our study analyzed the effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with Dengue virus. In presence of BaltPLA2, the viability of infected cells increased significantly in virucidal, post-treatment, and adsorption assays. Although preliminary these results reveal the need for further studies to investigated whether BaltPLA2 has antiviral activity against Dengue virus.

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (Bothrops moojeni).

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Antineoplastic properties and pharmacological applications of Crotalus durissus terrificus snake venom.

Snake toxins are widely studied owing to their importance in snakebite accidents, a serious public health issue in tropical countries, and their broad therapeutic potential. Isolated fractions from venom produced by snakes of the genus Crotalus sp. present a wide variety of pharmacological uses such as antifungal, antiviral, antibacterial, and antitumor properties, among other therapeutic potentialities. Given the direct effect of this venom on tumor cells, isolation of its compounds is important for the characterization of its anticarcinogenic actions. Crotalus durissus terrificus venom and its toxins have been widely evaluated as potential candidates for the development of new antineoplastic therapies that are efficient against different tumor lines and cellular targets. This review highlights the venom toxins of this species, with a focus on their antineoplastic properties.

BmooMPα-I, a Metalloproteinase Isolated from Bothrops moojeni Venom, Reduces Blood Pressure, Reverses Left Ventricular Remodeling and Improves Cardiac Electrical Conduction in Rats with Renovascular Hypertension.

BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.

Platelet depletion enhances lethal, hemorrhagic and myotoxic activities of Bothrops asper snake venom in a murine model.

Platelets play key roles in hemostasis, inflammation, immune response, and tissue repair. Although it is known that viperid snake venoms induce thrombocytopenia and platelet hypoaggregation, the roles of these effects in the overall outcome of envenoming are poorly known. This study aimed to assess the effect of platelet depletion on several toxic activities induced by the venom of the Central American viperid snake Bothrops asper in a mouse model. A profound thrombocytopenia was induced in mice by the administration of aspercetin, a C-type lectin-like protein that induces platelet agglutination and drop in platelet counts, while a control group was treated with saline solution instead. Upon envenoming, animals rendered thrombocytopenic developed a higher extent of local and systemic hemorrhage and local myonecrosis, as compared to control envenomed mice. In addition, the median lethal dose (LD50), determined by the intraperitoneal route, was significantly lower in thrombocytopenic mice, underscoring a higher toxicity of venom in these conditions. No difference in the value of LD50 between the two groups was observed when using the intravenous route of injection, and no difference was observed in the magnitude and time-course of footpad edema. Skeletal muscle regeneration was assessed 14 days after venom injection in muscle. Both experimental groups showed a similarly poor regeneration, suggesting that platelets do not play a key role in the regenerative process in these experimental conditions. Results indicate that depletion of platelets increases hemorrhagic and myotoxic effects, as well as overall toxicity, of B. asper venom, implying that platelets play a protective hemostatic role in this model of envenoming.

Effects of cilostazol, a Phosphodiesterase-3 inhibitor, on kidney function and redox imbalance in acute kidney injury caused by Bothrops alternatus venom.

The mechanisms of pathogenesis of acute kidney injury (AKI) in snakebites is multifactorial and involves hemodynamic disturbances, with release of free radical causing cytotoxic effects. The phosphodiesterase-3 (PDE3) inhibitor, Cilostazol, has been reported to provide protection against renal oxidative stress. OBJECTIVE: We evaluated the protective effects of cilostazol against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity. METHODS: Wistar rat kidneys (n = 6, 260-300 g) were isolated and perfused with Krebs-Henseleit solution containing 6 g/100 mL of bovine serum albumin. After 30 min, the kidneys were perfused with BaV to a final concentration of 1 and 3 µg/mL, and subsequently evaluated for perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl-). Oxidative stress and renal histological analyses were performed. RESULTS: BaV caused a reduction in all the evaluated renal parameters (PP, RVR, GFR, UF, %TNa+, and %TCl-). Although only the effects on PP and UF were reversed with cilostazol treatment, the decrease in the malondialdehyde levels, without changes in glutathione levels, further reduced the venom-induced renal tissue changes. CONCLUSION: Our data suggest that PDE3 is involved in BaV-induced nephrotoxicity, as cilostazol administration significantly ameliorated these effects.

Light Emitting Diode Photobiomodulation Enhances Oxidative Redox Capacity in Murine Macrophages Stimulated with Bothrops jararacussu Venom and Isolated PLA2s.

Photobiomodulation therapy associated with conventional antivenom treatment has been shown to be effective in reducing the local effects caused by bothropic venoms in preclinical studies. In this study, we analyzed the influence of photobiomodulation using light emitting diode (LED) on the oxidative stress produced by murine macrophages stimulated with Bothrops jararacussu venom and it isolated toxins BthTX-I and BthTX-II. Under LED treatment, we evaluated the activity of the antioxidant enzymes catalase, superoxide dismutase, and peroxidase as well as the release of hydrogen peroxide and the enzyme lactate dehydrogenase. To investigate whether NADPH oxidase complex activation and mitochondrial pathways could contribute to hydrogen peroxide production by macrophages, we tested the effect of two selective inhibitors, apocynin and CCCP3, respectively. Our results showed that LED therapy was able to decrease the production of hydrogen peroxide and the liberation of lactate dehydrogenase, indicating less cell damage. In addition, the antioxidant enzymes catalase, superoxide dismutase, and peroxidase increased in response to LED treatment. The effect of LED treatment on macrophages was inhibited by CCCP3, but not by apocynin. These findings show that LED photobiomodulation treatment protects macrophages, at least in part, by reducing oxidative stress caused B. jararacussu venom and toxins.

The anti-ovarian carcinoma activity of L-amino acid oxidase from Crotalus adamanteus venom in vivo and in vitro.

Snake venom L-Amino-acid oxidase (svLAAO) has become a critical research target in molecular biology and medical science since its widespread presence and diverse biological roles, including antitumor application. Our research confirmed that Crotalus adamanteus (C. adamanteus) venom LAAO exhibited potential anti-ovarian cancer activity both in vivo and in vitro. C. adamanteus venom LAAO significantly reduced viability of ovarian cancer cells and caused morphological changes that preceded cell death. The results of molecular biology experiments showed that C. adamanteus venom LAAO caused expression changes of genes related to apoptotic pathways either intrinsically or extrinsically in ovarian cancer cells. Animal experiments and histological analysis also proved that C. adamanteus venom LAAO could effectively inhibit the tissue damage caused by ovarian cancer, and animals treated with C. adamanteus venom LAAO showed higher survival time. Catalase blocked the major apoptosis induction of C. adamanteus venom LAAO on ovarian cancer cells, suggesting that the cytotoxicity of C. adamanteus venom LAAO on ovarian cancer cells was mainly mediated by H2O2. These results infer that C. adamanteus venom LAAO will have some advantages in new drug research and antitumor drug development in future.

Effect of the phospholipase A2 inhibitor Varespladib, and its synergism with crotalic antivenom, on the neuromuscular blockade induced by Crotalus durissus terrificus venom (with and without crotamine) in mouse neuromuscular preparations.

The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A2 (PLA2) crotoxin. Some populations of this subspecies contain, in addition to crotoxin, the toxin crotamine, which acts directly on muscle fibers. In this study we used C. d. terrificus venoms with (crot+) or without (crot-) crotamine to test whether Varespladib, a PLA2 inhibitor, is able to abrogate the neuromuscular blockade induced by these venoms comparatively with crotalic antivenom. Mouse phrenic nerve-diaphragm preparations were exposed to venoms previously incubated with two different concentrations of Varepladib or antivenom, or with a mixture of these two agents, before addition to the bath. In another experimental setting, venoms were initially added to the system, followed by the addition of Varespladib or antivenom 10, 30, or 60 min after venom. At the highest concentrations tested, Varespladib and antivenom inhibited the action of the venom >80% and >70%, respectively. With lower concentrations the inhibition of neuromuscular blockade decreased, but when low doses of the two agents were incubated together with the venom, the inhibitory effect improved, underscoring a synergistic phenomenon. When added after venom, Varespladib was able to halt the progression of the neuromuscular blockade even when added at 60 min. Antivenom exhibited a lower ability to inhibit the toxic effect of the venoms in these conditions. In conclusion, the PLA2 inhibitor Varespladib is highly effective at abrogating the neuromuscular blocking activity of crotamine-positive and crotamine-negative C. d. terrificus venoms and seems to act synergistically with antivenom.

Evidence that BJcuL, a C-type lectin from Bothrops jararacussu venom, influences deubiquitinase activity, resulting in the accumulation of anti-apoptotic proteins in two colorectal cancer cell lines.

BJcuL is a snake venom C-type lectin (SVCTL) purified from the snake's venom Bothrops jararacussu. It has been previously demonstrated that BJcuL induces the accumulation of pro-apoptotic proteins of the extrinsic pathway, such as FADD and caspase-8, in the colorectal cancer cell line HT29, suggesting that the lectin may be able to enhance TRAIL-induced apoptosis. To test this hypothesis, we exposed two colorectal cancer cell lines, HT29 and HCT116, to increasing concentrations of BJcuL (1-20 µg/mL) in the presence or absence of TRAIL. Contrary to our expectations, however, BJcuL was unable to induce apoptosis in these cells, as shown by annexin-V/7AAD, clonogenic assays, and immunoblotting. Nevertheless, BJcuL was able to induce the accumulation of FADD and caspase-8, as well as anti-apoptotic proteins such as c-FLIP and survivin and poly-ubiquitinated proteins. Incubation with the deubiquitinase inhibitor WP1130 (10 µM) resulted in decreased BJcuL-induced survivin levels. Altogether, our results evince the effects of SVCTL on the ubiquitin-proteasome system in vitro for the first time. Compounds that can influence such system are important tools in the search for new therapeutic or diagnostic targets in cancer since they can elucidate the molecular mechanisms involved in determining cell fate as well as contributing to drug-development strategies in partnership with the pharmaceutical industry.

The Effects of αvß3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro.

Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvß3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvß3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvß3 integrin during tumor progression.

A New Approach to Inhibiting Triple-Negative Breast Cancer: In Vitro, Ex Vivo and In Vivo Antiangiogenic Effect of BthTx-II, a PLA2-Asp-49 from Bothrops jararacussu Venom.

Phospholipases A2 (PLA2) represent a superfamily of enzymes widely distributed in living organisms, with a broad spectrum of pharmacological activities and therapeutic potential. Anti-angiogenic strategies have become one of the main tools in fighting cancer. In this sense, the present work reports the inhibition of tumor angiogenesis induced by Asp-49 BthTX-II using in vitro, ex vivo and in vivo approaches. We demonstrate that BthTx-II inhibited cell adhesion, proliferation, and migration of human umbilical vein endothelial cells (HUVEC), as well as caused a reduction in the levels of endothelial growth factor (VEGF) during in vitro angiogenesis assays. BthTx-II was also able to inhibit the sprouting angiogenic process, by the ex vivo germination assay of the aortic ring; in addition, this toxin inhibited the migration and proliferation of HUVEC in co-culture with triple-negative breast cancer cells (e.g., MDA-MB-231 cells). Finally, in vivo tumor suppression and anti-angiogenic activities were analyzed using MDA-MB-231 cells with Matrigel injected into the chorioallantoic membrane of chicken embryo (CAM) for 7 days treatment with BthTx-II, showing a considerable reduction in vessel caliber, on the size and weight of tumors. Together, these results suggest an important antiangiogenic and antitumor role for BthTx-II, as a potential prototype for the development of new tools and antitumor drugs in cancer therapy.