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The aim of this study was to evaluate the effects of different doses of alternagin-C, a disintegrin-like protein from Rhinocerophis alternatus venom, on myocardial contractility of the freshwater fish Hoplias malabaricus, an alternative model to contractile function studies. Alternagin-C treatment exhibited a hormetic-like dose-response curve with a strong positive inotropism and enhanced cardiac pumping capacity at low dose, whereas a modest inotropism and a left shift in the force-frequency relationship was registered at high dose.

The incorporation of osteoconductive hydroxyapatite (HA) into poly(Ɛ-polycaprolactone) (PCL) may enhance the material hydrophilicity, protein adsorption, roughness, and consequently, bone formation. In this work, PCL/HA composites with 5, 10, and 25 wt% of HA were prepared by melt compounding followed by hot compression, and their properties such as torque, molecular weight, mechanical resistance, and viscosity were compared to neat PCL to understand the influence of the filler on the polymer stability and printability. The addition of 5 and 10 wt% of HA leads to properties similar to the neat PCL; therefore, these compositions were chosen to produce scaffolds by 3D printing. The scaffolds presented excellent printability and homogenous dispersion of the HA. The compressive strength modulus of both compressed samples and scaffolds is around 30 MPa, similar to cancellous bone. The presence of increasing HA content combined with surface treatment using NaOH enhanced osteoblast proliferation.

Angiogenesis, a crucial process in tumor progression, is mainly regulated by vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. Studies have shown the interaction between α2β1 integrin, a collagen receptor, and VEGFR2 in VEGF-driven angiogenesis in vitro and in vivo. Alternagin-C (ALT-C), an ECD-disintegrin-like protein from Bothrops alternatus snake venom, has high affinity for α2β1 integrin and shows antiangiogenic activity in concentrations higher than 100 nM. Despite previous results, its mechanism of action on angiogenic signaling pathways has not been addressed. Here we evaluate the antiangiogenic activity of ALT-C in human umbilical vein endothelial cells (HUVECs) associated or not with VEGF, as well as its interference in the α2β1/VEGFR2 crosstalk. ALT-C (1000 nM) affected actin cytoskeleton, decreased the number of cell filopodia, and strongly inhibited HUVEC tube formation, adhesion to type I collagen and cell migration. Down-regulation of α2β1/VEGFR2 crosstalk by ALT-C decreased the protein content and phosphorylation of VEGFR2 and β1 integrin subunit, inhibited ERK 1/2 and PI3K signaling and regulated FAK/Src and paxillin pathways. Furthermore, ALT-C increased the content of the autophagic markers LC3B and Beclin-1 in the presence of VEGF, which is associated with decreased angiogenesis. In conclusion, we suggest that ALT-C, after binding to α2β1 integrin, inhibits VEGF/VEGFR2 signaling, which results in impaired angiogenesis. These results demonstrate that ALT-C may be a potential candidate for the development of antiangiogenic therapies for tumor and metastasis treatment and help to understand the complexity and fundamental role of integrin inhibition in the tumor microenvironment.

Photodynamic Therapy (PDT) is a promising treatment for various types of cancer and other non-oncological diseases. This technique uses a photosensitizer (PS) that generates reactive oxygen species (ROS), which lead cells to death in the presence of light and molecular oxygen. In this study, we evaluated the photoactivity of four chlorins that presented an L-type structure conformation (6a, 6b, 8a, and 8b) in two tumoral (HEp-2 and HeLa) and one non-tumoral (Vero) cell line under PDT conditions. Intracellular accumulation and subcellular localization were determined by confocal microscopy, showing that these chlorins exhibited an excellent (120 min) internalization through the plasmatic membrane with mitochondria and lysosome localization. The chlorins 6a-b were two and three-fold more selective than the chlorins 8a-b to HEp-2 and HeLa compared to Vero at 3 and 6 J cm-2 doses, respectively. The fluorescence microscopy and flow cytometry analysis showed that the four chlorin derivatives lead tumor cells to death predominantly by apoptosis during the treatment with light. Considering that the chlorins 6a-b exhibited good water solubility and high phototoxicity to the tumor cells, we suggested that these tetrapyrrolic molecules have the potential for clinical treatment in cancer by PDT.

Human epidermal growth factor receptor-2 (HER2)-targeting therapies provide clinical benefits for patients with HER2-positive breast cancer. However, the resistance to monotherapies invariably develops and leads to disease relapse and treatment failure. Previous studies have demonstrated a link between the potency of HER2-targeting tyrosine kinase inhibitors (TKIs) and their ability to induce an iron-dependent form of cell death called ferroptosis. The aim of this study was to understand the mechanisms of resistance to TKI-induced ferroptosis and identify novel approaches to overcome treatment resistance. We used mouse and human HER2-positive models of acquired TKI resistance to demonstrate an intimate link between the resistance to TKIs and to ferroptosis and present the first evidence that the cell adhesion receptor αvβ3 integrin is a critical mediator of resistance to TKI-induced ferroptosis. Our findings indicate that αvβ3 integrin-mediated resistance is associated with the re-wiring of the iron/antioxidant metabolism and persistent activation of AKT signalling. Moreover, using gene manipulation approaches and pharmacological inhibitors, we show that this “αvβ3 integrin addiction” can be targeted to reverse TKI resistance. Collectively, these findings provide critical insights into new therapeutic strategies to improve the treatment of advanced HER2-positive breast cancer patients.

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.

The spread of cells from primary tumors toward distant tissues and organs, also known as metastasis, is responsible for most cancer-associated deaths. The metastasis cascade comprises a series of events, characterized by the displacement of tumor cells (TCs) from the primary tumor to distant organs by traveling through the bloodstream, and their subsequent colonization. The first step in metastasis involves loss of cell-cell and cell-matrix adhesions, increased invasiveness and migratory abilities, leading to intravasation of TCs into the blood or lymphatic vessels. Stationary TCs must undergo the process of epithelial-mesenchymal transition in order to achieve this migratory and invasive phenotype. Circulating tumor cells that have survived in the circulation and left the blood or lymphatic vessels will reach distant sites where they may stay dormant for many years or grow to form secondary tumors. To do this, cells need to go through the mesenchymal-epithelial transition to revert the phenotype in order to regain epithelial cell-to-cell junctions, grow and become a clinically relevant and detectable tumor mass. This work will review the main steps of the metastatic cascade and describe some strategies to inhibit metastasis by reducing cancer cell extravasation presenting recent studies in the context of breast cancer.

Background Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvβ3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αvβ3 integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αvβ3/VEGFR2 cross-talk and its downstream pathways. Methods Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αvβ3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy. Results DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvβ3/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and β3 integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αvβ3 integrin and VEGFR2 in treated cells. Conclusions Disintegrin inhibition of αvβ3 integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis. Electronic supplementary material The online version of this article (10.1186/s12964-019-0339-1) contains supplementary material, which is available to authorized users.

Made available in DSpace on 2021-06-25T12:41:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) National Science Foundation Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle. Rensselaer Polytech Inst, Dept Chem & Biol Engn, Troy, NY 12180 USA Rensselaer Polytech Inst, Dept Biol Sci, Troy, NY 12180 USA Rensselaer Polytech Inst, Ctr Biotechnol & Interdisciplinary Studies, Troy, NY USA Univ Fed Sao Carlos, Dept Chem Engn, BR-13565905 Sao Carlos, SP, Brazil Sao Paulo State Univ, Ctr Study Social Insects, BR-13506900 Rio Claro, SP, Brazil Univ Fed Sao Carlos, Dept Physiol Sci, BR-13565905 Sao Carlos, SP, Brazil Sao Paulo State Univ, Ctr Study Social Insects, BR-13506900 Rio Claro, SP, Brazil FAPESP: FAPESP 2017/09695-2 CAPES: 001 National Science Foundation: NSF-1616674 FAPESP: 2019/11437-7

Thirteen new ruthenium amino acid complexes were synthesized and characterized. They were obtained by the reaction of α-amino acids (AA) with [RuCl2(P-P)(N-N)], where P-P=1,4-bis(diphenylphosphino)butane (dppb) or 1,3-bis(diphenylphosphino)propane (dppp) and N-N=4,4'-dimethyl-2,2'-bipyridine (4'-Mebipy), 5,5'-dimethyl-2,2'-bipyridine (5'-Mebipy) or 4,4'-Methoxy-2-2'-bipyridine (4'-MeObipy). This afforded a family of complexes formulated as [Ru(AA-H)(P-P)(N-N)]PF6, where AA=glycine (Gly), L-alanine (Ala), L-valine (Val), L-tyrosine (Tyr), L-tryptophan (Trp), L-histidine (His) and L-methionine (Met). All compounds were characterized by elemental analysis, spectroscopic and electrochemical techniques. The [Ru(AA-H)(P-P)(N-N)]PF6 complexes are octahedral (the AA-H ligand binding involves N-amine and O-carboxylate), diamagnetic (low-spin d6, S=0) and present bands due to electronic transitions in the visible region. 1H, 13C{1H} and 31P{1H} NMR spectra of the complexes indicate the presence of C2 symmetry, and the identification of diastereoisomers. In vitro cytotoxicity assays of the compounds and cisplatin were carried out using MDA-MB-231 (human breast) tumor cell line and a non-tumor breast cell line (MCF-10A). Most complexes present promising results with IC50 values comparable with the reference drug cisplatin and high selectivity indexes were found for the complexes containing L-Trp. The binding of two Ru-precursors of the type [RuCl2(dppb)(NN)] (N-N=4'-MeObipy or 4'-Mebipy) to the blood transporter protein human serum albumin (HSA) was evaluated by fluorescence and circular dichroism spectroscopy. Both complexes bind HSA, probably in the hydrophobic pocket near Trp214, and the Ru-complex containing 4'-MeObipy shows higher affinity for HSA than the 4'-Mebipy one.

The calcaneal tendon (CT) is the most commonly injured tendon in the human body. Moreover, with advancing age, the amount of damage increases further. Resistance training (RT) could be used to minimize such damages. The aim of the present study was to obtain the identification, detailed protein cataloging and biochemical characterization based on the effects of the aging process and the RT in CT of rats. The analysis by liquid chromatography tandem-mass spectrometry showed 142 distinct proteins, however, only 29 proteins met the inclusion criteria and were analyzed. Aging causes a reduction in the abundance of seven proteins related to extracellular matrix organization, biological regulation and cellular processes. However, RT promoted the positive regulation of proteins important for the maintenance of healthy tendons: seven proteins in young trained and two in older trained group. This study contributes to a better understanding of molecular aspects of the tendon. The down regulation of proteins linked to mechanical function and extracellular matrix remodeling of the tendon during aging can contribute to the increase of injury and weakness in the tendon. Nevertheless, RT proved to be a tool to prevent these adverse effects during aging by increasing proteins involved in the functionality of the tendon.

Sarcopenia is a complex multifactorial process, some of which involves fat infiltration. Intramyocellular lipid (IMCL) accumulation is postulated to play a role on sarcopenia during aging, which is believed to be due alterations in glucose homeostasis in the skeletal muscle. Sarcopenia, along with intramuscular lipids, is associated with physical inactivity. Resistance training (RT) has been indicated to minimize the age-induced muscle skeletal adaptations. Thus, we aimed to investigate the effects of RT on mRNA levels of regulatory components related to intramyocellular lipid, glucose metabolism and fiber size in soleus and gastrocnemius muscles of aged rats. Old male rats were submitted to RT (ladder climbing, progressive load, 3 times a week for 12 weeks). Age-induced accumulation of IMCL was attenuated by RT, which was linked to a PPARy-mediated mechanism, concomitant to enhanced regulatory components of glucose homeostasis (GLUT-4, G6PDH, Hk-2 and Gly-Syn-1). These responses were also linked to decreased catabolic (TNF-α, TWEAK/Fn14 axis; FOXO-1, Atrogin-1 and MuRF1; Myostatin) and increased anabolic intracellular pathways (IGF-1-mTOR-p70S6sk-1 axis; MyoD) in muscles of trained aged rats. Our results point out the importance of RT on modulation of gene expression of intracellular regulators related to age-induced morphological and metabolic adaptations in skeletal muscle.

Made available in DSpace on 2018-12-11T17:11:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Six new complexes of Ru(II)/phenanthroline/1,4-bis(diphenylphosphino)butane containing amino acids (Glycine, L-Alanine, L-Valine, L-Tyrosine, L-Methionine or L-Tryptophan) were synthesized and characterized by IR, 31P{1H}, 13C and 1H NMR spectroscopies and cyclic voltammetry experiments. These data suggest the presence of diastereoisomers, except for the complex with glycine, amino acid that does not exhibit chiral carbon. The compounds are active against the MDA-MB-231 tumor cells and against Mycobacterium tuberculosis. The cationic ruthenium complexes with amino acids, reported here, show similar cytotoxicity against the MDA-MB-231 tumor cells. When compared with analogs complexes containing 2,2′-bipyridine as ligands, instead of 1,10-phenatroline, the new complexes studied here are, in general, roughly twice more active than the 2,2′-bipyridine ones and their IC50 values comparable with the cisplatin. In addition, low MICs values were obtained against Mycobacterium tuberculosis compared with the reference drugs, cycloserine and ethambutol. Departamento de Química Universidade Federal de São Carlos, C.P. 676 Departamento de Química ICEB Universidade Federal de Ouro Preto Departamento de Ciências Fisiológicas Universidade Federal de São Carlos, C.P. 676 Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas UNESP Departamento de Ciências Biológicas Faculdade de Ciências Farmacêuticas UNESP

Purpose Verify if sodium nitroprusside (SNP) is able to improve endothelial function and if this effect is independent of nitric oxide (NO) release of the compound. Methods Normotensive (2K) and hypertensive (2K-1C) wistar rats were used. Intact endothelium aortas were placed in a myograph and incubated with SNP: 0.1 nM; 1 nM or 10 nM during 30 min. Cumulative concentration-effect curves for acetylcholine (Ach) were realized to measure the relaxing capacity. Intracellular NO were measured (by DAF-2DA probe) in HUVEC treated with SNP 0.1 nM or DETA/NO 0.1 μM. The detection of intracellular superoxide radical (O 2 •- ) was obtained by using DHE probe. Results Treatment of 2K-1C aortic rings with SNP (0.1; 1.0 and 10 nM) improved endothelium dependent relaxation induced by acetylcholine. This improvement induced by SNP was verified at the concentration of 0.1 nM, which does not release NO, suggesting that this effect was not induced due to NO release by SNP compound. Besides, we show that the cell treatment with 0.1 nM of SNP decreased the fluorescence intensity to DHE in cells stimulated with angiotensin II. These results indicate that SNP decreases the concentration of O 2 •- in HUVEC cells. Conclusions The SNP at a concentration that does not release NO inside the cells is able to attenuate endothelial dysfunction. Drugs and chemicals Acetylcholine (Ach) (PubChem CID:6060); angiotensin II human (Ang II) (PubChem CID: 16211177); diethylenetriamine/nitric oxide (DETA-NO) (PubChem CID 4518); dihydroethidium (DHE) (PubChem CID: 128682); phenylephrine (Phe) (PubChem CID: 5284443); sodium nitroprusside (SNP) (PubChem CID: 11963579); Thiazolyl Blue Tetrazolium Bromide (MTT) (PubChem CID: 64965); 4,5-diaminofluorescein diacetate (DAF-2DA); 4-hidroxy-Tempo (Tempol) (PubChem CID: 137994), were purchased from Sigma–Aldrich (St. Louis, MO, USA).

Doxorubicin (Doxo), cisplatin (Cis), and tamoxifen (Tamo) are part of many chemotherapeutic regimens. However, there have been limited studies of the way metabolism in breast cancer is affected by chemotherapy. We studied, through 1H high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy, the metabolic profile of human breast cancer MCF-7 control (Con) cells as well as MCF-7 cells treated with Tamo, Cis, and Doxo. 1H HR-MAS NMR single-pulse spectra evidenced signals from the cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites. The spectra showed that phosphocholine (i.e., biomarker of breast cancer malignant transformation) signals were stronger in Con than in treated cells. Betaine (i.e., the major osmolyte in cells) was observed at similar concentrations in MCF-7 control and treated cells but was absent in nontumor MCF-10A cells. The NMR spectra acquired with the Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence were used only in qu...

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.

Aging process is characterized by a decline in the organism functionality, especially in the decrease of muscle function, which also affects tendons. On the other hand, the resistance training (RT) has been used as an important tool to increase muscle and tendineous function during aging. Thus, this study aim has been to verify the effects of RT on the biomechanical properties of three different aged rat tendons. For this purpose, 20 wistar rats have been divided into four groups (5 rats per group): young sedentary (YS), trained (YT), old sedentary (OS) and old trained (OT). The RT has been performed through climb protocol for 12 weeks. After RT, the calcaneal tendon (CT), superficial flexor tendon (SFT) and deep flexor tendon (DFT) have been used for analysis. The results indicate that the RT in aged rats can prevent tendon function decrease (p

Sarcopenia is a complex multifactorial process, some of which involves fat infiltration. Intramyocellular lipid (IMCL) accumulation is postulated to play a role on sarcopenia during aging, which is believed to be due alterations in glucose homeostasis in the skeletal muscle. Sarcopenia, along with intramuscular lipids, is associated with physical inactivity. Resistance training (RT) has been indicated to minimize the age-induced muscle skeletal adaptations. Thus, we aimed to investigate the effects of RT on mRNA levels of regulatory components related to intramyocellular lipid, glucose metabolism and fiber size in soleus and gastrocnemius muscles of aged rats. Old male rats were submitted to RT (ladder climbing, progressive load, 3 times a week for 12 weeks). Age-induced accumulation of IMCL was attenuated by RT, which was linked to a PPARy-mediated mechanism, concomitant to enhanced regulatory components of glucose homeostasis (GLUT-4, G6PDH, Hk-2 and Gly-Syn-1). These responses were also linked to decreased catabolic (TNF-α, TWEAK/Fn14 axis; FOXO-1, Atrogin-1 and MuRF1; Myostatin) and increased anabolic intracellular pathways (IGF-1-mTOR-p70S6sk-1 axis; MyoD) in muscles of trained aged rats. Our results point out the importance of RT on modulation of gene expression of intracellular regulators related to age-induced morphological and metabolic adaptations in skeletal muscle.

Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The αvβ3 integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of αvβ3 integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, αvβ3 integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.

Scaffolds of poly(ε-caprolactone) (PCL) and their biocomposites with 0, 1, 3, and 5 wt.% Biosilicate® were fabricated by the generative manufacturing process coupled with a vertical miniscrew extrusion head to application for restoration of bone tissue. Their morphological characterization indicated the designed 0°/90° architecture range of pore sizes and their interconnectivity is feasible for tissue engineering applications. Mechanical compression tests revealed an up to 57% increase in the stiffness of the scaffold structures with the addition of 1 to 5 wt.% Biosilicate® to the biocomposite. No toxicity was detected in the scaffolds tested by in vitro cell viability with MC3T3-E1 preosteoblast cell line. The results highlighted the potential application of scaffolds fabricated with poly(ε-caprolactone)/Biosilicate® to tissue engineering.

Exercise training (ET), anabolic androgenic steroids (AAS), and aging are potential factors that affect tendon homeostasis, particularly extracellular matrix (ECM) remodeling. The goal of this review is to aggregate findings regarding the effects of resistance training (RT), AAS, and aging on tendon homeostasis. Data were gathered from our studies regarding the impact of RT, AAS, and aging on the calcaneal tendon (CT) of rats. We demonstrated a series of detrimental effects of AAS and aging on functional and biomechanical parameters, including the volume density of blood vessel cells, adipose tissue cells, tendon calcification, collagen content, the regulation of the major proteins related to the metabolic/development processes of tendons, and ECM remodeling. Conversely, RT seems to mitigate age-related tendon dysfunction. Our results suggest that AAS combined with high-intensity RT exert harmful effects on ECM remodeling, and also instigate molecular and biomechanical adaptations in the CT. Moreover, we provide further information regarding the harmful effects of AAS on tendons at a transcriptional level, and demonstrate the beneficial effects of RT against the age-induced tendon adaptations of rats. Our studies might contribute in terms of clinical approaches in favor of the benefits of ET against tendinopathy conditions, and provide a warning on the harmful effects of the misuse of AAS on tendon development.

The increase of tumor mass and complexity triggers cell invasion, a key step of the metastatic cascade, through hypoxia-derived signalization. The decrease in oxygen levels on the inner tumor mass stimulates the secretion of extracellular vesicles (EV) to the microenvironment, whose cargo may induce an invasive behavior of breast cancer cells. In an attempt to identify key players in hypoxic EV-mediated cell invasion in breast cancer, we are investigating the molecular, cellular and biochemical changes triggered by hypoxic-EVs to triple-negative breast cancer cells (MDA-MB-231). EVs have been isolated from conditioned culture media by differential ultracentrifugation and characterized by particle analysis (NTA), transmission electron microscopy (TEM), protein quantification and western blot of EV markers according to the MISEV2018. EV-treated cells were submitted to invasion assays in i) invasion chamber with extracellular matrix-based hydrogel and ii) a fluorescent gelatin-coated surface. Cell lysate and EV samples were probed for matrix metalloproteases by western blot and zymography. Morphological analysis of cells after EV treatment have been conducted after cytoskeleton staining using fluorescent probes. EVs derived from hypoxia and normoxia-cultured MDA-MB-231 are similarly sized and enriched with CD63, FLOT-1 and ALIX, although hypoxic-EVs have displayed higher protein concentration and particle concentration. Hypoxic EVs promoted cell invasion in an extracellular matrix-based hydrogel after 6h and 16h under normal oxygen conditions. Gelatin degradation has also been observed after EV treatment in normoxia, with results akin to degradation found in cells under hypoxia. While protease activity has been detected in EVs via zymography, we have yet to confirm the role of matrix metalloproteases in EV-related cell invasion. While little morphological changes have been observed upon EV treatment in gelatin-adhered cells, they acquire an epithelial morphology in uncoated circumstances, thus indicating a key role of adhesion molecules to hypoxia-derived invasive behavior. In conclusion, hypoxia-derived EVs from breast cancer cells generate morphological and physiological changes to tumor cells in regular oxygen levels, leading to an invasive behavior in collagen-based matrixes. Citation Format: Bianca Cruz Pachane, Wanessa Fernanda Altei, Heloisa Sobreiro Selistre-de-Araujo. Hypoxia-derived extracellular vesicles promote triple-negative breast cancer invasion in vitro [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO034.

We herein report the synthesis and characterization of Ru(II)/amino acid complexes with general formula [Ru(AA-H)(dppb)(4-mebipy)](PF6), where AA-H means the deprotonated amino acids Gly, Ala, Val, Met, Trp, Tyr, and Ser; dppb is 1,4-bis(diphenylphosphino)butane and 4-mebipy = 4,4′-dimethyl-2,2′-bipyridine. The complexes were characterized by 31P{1H}, 13C, and 1H NMR spectroscopy, as well as X-ray crystallographic analysis of [Ru(DL-Ala-H)(dppb)(4-mebipy)]+, suggesting the presence of diastereoisomers. The complexes exhibit IC50 values against breast tumor cells (MDA-MB-231) comparable with cisplatin. In addition, the Ru(II)-based complex with tryptophan inhibited tumor cell adhesion to collagen type I. Therefore, the use of ruthenium complexes containing amino acids can be an interesting tool for development of new therapeutic agents.

OBJECTIVE: The purpose of this study was to investigate the effects of resistance training on angiogenesis markers of visceral adipose tissue in ovariectomized rats. METHOD: Adult Sprague-Dawley female rats were divided into four groups (n=6 per group): sham-sedentary, ovariectomized sedentary, sham-resistance training and ovariectomized resistance training. The rats were allowed to climb a 1.1-m vertical ladder with weights attached to their tails and the weights were progressively increased. Sessions were performed three times per week for 10 weeks. Visceral adipose tissue angiogenesis and morphology were analyzed by histology. VEGF-A mRNA and protein levels were analyzed by real-time PCR and ELISA, respectively. RESULTS: Ovariectomy resulted in higher body mass (p=0.0003), adipocyte hypertrophy (p=0.0003), decreased VEGF-A mRNA (p=0.0004) and protein levels (p=0.0009), and decreased micro-vascular density (p=0.0181) in the visceral adipose tissue of the rats. Resistance training for 10 weeks was not able to attenuate the reduced angiogenesis in the visceral adipose tissue of the ovariectomized rats. CONCLUSION: Our findings indicate that the resistance training program used in this study could not ameliorate low angiogenesis in the visceral adipose tissue of ovariectomized rats.

Alternagin-C (ALT-C) is a disintegrin-like protein purified from the venom of the snake, Rhinocerophis alternatus. Recent studies showed that ALT-C is able to induce vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, angiogenesis and to increase myoblast viability. This peptide, therefore, can play a crucial role in tissue regeneration mechanisms. The aim of this study was to evaluate the effects of a single dose of alternagin-C (0.5 mg kg(-1), via intra-arterial) on in vitro cardiac function of the freshwater fish traíra, Hoplias malabaricus, after 7 days. ALT-C treatment increased the cardiac performance promoting: 1) significant increases in the contraction force and in the rates of contraction and relaxation with concomitant decreases in the values of time to the peak tension and time to half- and 90% relaxation; 2) improvement in the cardiac pumping capacity and maximal electrical stimulation frequency, shifting the optimum frequency curve upward and to the right; 3) increases in myocardial VEGF levels and expression of key Ca(2+)-cycling proteins such as SERCA (sarcoplasmic reticulum Ca(2+)-ATPase), PLB (phospholamban), and NCX (Na(+)/Ca(2+) exchanger); 4) abolishment of the typical negative force-frequency relationship of fish myocardium. In conclusion, this study indicates that ALT-C improves cardiac function, by increasing Ca(2+) handling efficiency leading to a positive inotropism and chronotropism. The results suggest that ALT-C may lead to better cardiac output regulation indicating its potential application in therapies for cardiac contractile dysfunction.

Several side effects of anabolic-androgenic steroid (AAS) administration associated with training are reported in the biomechanical properties of the calcaneal tendon (CT) of rats. Thus, the aim of the present study is to evaluate the effects of the detraining and discontinuation of AAS administration on the CT morphology of rats submitted to exercise in water. Animals were divided into two groups (20/group): (1) Immediately after training (IA), and (2) Six weeks of detraining and AAS discontinuation (6W). The IA group included four subgroups: Sedentary (S), Trained (T), Sedentary with AAS administration (SAAS), and trained with AAS administration (TAAS). The 6W group included four subgroups: Sedentary (6W-S), six weeks of detrained (6W-T), six weeks of sedentary with AAS discontinuation (6W-SAAS), and six weeks of detrained with AAS discontinuation (6W-TAAS). Data show significant reduction in adipose cells volume density (Vv%) in the distal CT in 6W-TAAS group, indicating that training can exert a positive effect on the tendon. The 6W-SAAS group exhibited increased adipose cells Vv% in the distal region, compared with the W6-S and W6-T groups. A decrease in tendon proper cells Vv% and in peritendinous sheath cells Vv% of proximal and distal regions was also observed. In 6W-TAAS group showed increase in adipose cells, blood vessels, peritendinous sheath cells, and tendon proper cells Vv% in the distal region of the CT. The vertical jumps in water were not able to protect CT regions from the negative effects of AAS discontinuation for six weeks. However, after detraining and AAS discontinuation, many protective factors of the mechanical load in the long-term could be observed.

Alternagin-C (ALT-C) is a disintegrin-like peptide purified from Rhinocerophis alternatus snake venom with the property of inducing vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, and angiogenesis. Therefore, this protein could be interesting as a new approach for ischemic heart diseases, an imbalance between myocardial oxygen supply and demand, leading to cardiac dysfunction. We investigated the effects of a single dose of alternagin-C (0.5 mg kg−1, via intra-arterial), after 7 days, on hypoxia/reoxygenation challenge in isolated ventricle strips and on morphological changes and density of blood vessels of the heart, using fish as an alternative experimental model. ALT-C treatment provided protection of cardiomyocytes against hypoxia/reoxygenation-induced negative inotropism. ALT-C also stimulated angiogenesis and improved excitation-contraction coupling during hypoxic conditions. Our results provide a new insight into a functional role of ALT-C against hypoxia/reoxygenation-induced cardiomyocyte injury pointing out to a potential therapeutic strategy for ischemia-related diseases.

Accumulation of connective tissue, particularly extracellular matrix (ECM) proteins, has been observed in skeletal muscles with advancing age. Resistance training (RT) has been widely recommended to attenuate age-induced sarcopenia, even though its effects on the components that control ECM turnover in skeletal muscles remain to be elucidated. Thus, the aim of this study was to determine the effects of RT on connective tissue content and gene expression of key components of ECM in the skeletal muscles of aged rats. Young (3 mo.) and older (21 mo.) adult male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), 3 times a week for 12 weeks. Forty-eight hours post-training, the soleus (SOL) and gastrocnemius (GAS) muscles were dissected for histological and mRNA analysis. RT mitigated the age-associated increase of connective tissue content in both muscles, even though mRNA levels of COL-1 and−3 were elevated in older trained rats. Overall, RT significantly elevated the gene expression of key components of connective tissue deposition (TGFβ and CTGF; MMP-2 and-9; TIMP-1 and−2) in the GAS and SOL muscles of older rats. In conclusion, RT blunted the age-induced accumulation of connective tissue concomitant to the upregulation of genes related to synthesis and degradation of the ECM network in the SOL and GAS muscles of older rats. Although our findings indicate that RT plays a crucial role reducing connective tissue accumulation in aged hindlimb muscles, key components of ECM turnover were paradoxically elevated. The phenotypic responses induced by RT were not accompanied by the gene expression of those components related to ECM turnover.

Background/objectivesRegular exercise training is effective to altering many markers of metabolic syndrome and its effects are strongly influenced by the type of consumed diet. Nowadays, resistance training (RT) has been frequently associated with low-carbohydrate high-fat diet (LCD). After long term these diets causes body weight (BW) regain with deleterious effects on body composition and metabolic risk factors. The effects of RT associated with long-term LCD on these parameters remain unexplored. We aimed to investigate the effects of RT when associated with long-term LCD on BW, feed efficiency, body composition, glucose homeostasis, liver parameters and serum biochemical parameters during BW regain period in rats.Subjects/methodsMale Sprague-Dawley rats were fed with LCD (LC groups) or standard diet (STD) (ST groups). After 10 weeks-diet animals were separated into sedentary (Sed-LC and Sed-ST) and resistance-trained (RT-LC and RT-ST) groups (N = 8/group). RT groups performed an 11-week climbing program on a ladder with progressive load. Dual x-ray absorptiometry, glucose tolerance tests and insulin tolerance tests were performed at weeks 10 and 20. Liver and serum were collected at week 21.ResultsRT reduced feed efficiency, BW gain, liver fat and total and LDL cholesterol, and improved body composition and glucose clearance in animals fed on STD. In those fed with LCD, RT reduced caloric intake, BW regain, liver fat and serum triglycerides levels. However, improvement in body composition was inhibited and bone mineral density and glucose clearance was further impaired in this association.ConclusionsThe LCD nullifies the beneficial effects of RT on body composition, glucose homeostasis and impairs some health parameters. Our results do not support the association of RT with LCD in a long term period.

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2β1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2β1 integrin. Herein, we used ALT-C as a α2β1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2β1 integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to α2β1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. Conclusion These results demonstrate that α2β1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies. Electronic supplementary material The online version of this article (10.1186/s40409-018-0150-2) contains supplementary material, which is available to authorized users.

One of the most important features of malignant cells is their capacity to invade adjacent tissues and metastasize to distant organs. This process involves the creation, by tumor and stroma cells, of a specific microenvironment, suitable for proliferation, migration and invasion of tumor cells. The ADAM family of proteins has been involved in these processes. This work aimed to investigate the role of the recombinant disintegrin domain of the human ADAM9 (rADAM9D) on the adhesive and mobility properties of DU145 prostate tumor cells. rADAM9D was able to support DU145 cell adhesion, inhibit the migration of DU145 cells, as well as the invasion of this cell line through matrigel in vitro. Overall this work demonstrates that rADAM9D induces specific cellular migratory properties when compared with different constructs having additional domains, specially those of metalloproteinase and cysteine-rich domains. Furthermore, we showed that rADAM9D was able to inhibit cell adhesion, migration and invasion mainly through interacting with α6β1 in DU145 tumor cell line. These results may contribute to the development of new therapeutic strategies for prostate cancer.

Although many preclinical studies have implicated β3 integrin receptors (αvβ3 and αIIbβ3) in cancer progression, β3 inhibitors have shown only modest efficacy in patients with advanced solid tumours. The limited efficacy of β3 inhibitors in patients could arise from our incomplete understanding of the precise function of β3 integrin and, consequently, inappropriate clinical application. Data from animal studies are conflicting and indicate heterogeneity with respect to the relative contributions of β3-expressing tumour and stromal cell populations in different cancers. Here we aimed to clarify the function and relative contributions to metastasis of tumour versus stromal β3 integrin in clinically relevant models of spontaneous breast cancer metastasis, with particular emphasis on bone metastasis. We show that stable down-regulation of tumour β3 integrin dramatically impairs spontaneous (but not experimental) metastasis to bone and lung without affecting primary tumour growth in the mammary gland. Unexpectedly, and in contrast to subcutaneous tumours, orthotopic tumour vascularity, growth and spontaneous metastasis were not altered in mice null for β3 integrin. Tumour β3 integrin promoted migration, protease expression and trans-endothelial migration in vitro and increased vascular dissemination in vivo, but was not necessary for bone colonization in experimental metastasis assays. We conclude that tumour, rather than stromal, β3 expression is essential and is required early for efficient spontaneous breast cancer metastasis to bone and soft tissues. Accordingly, differential gene expression analysis in cohorts of breast cancer patients showed a strong association between high β3 expression, early metastasis and shorter disease-free survival in patients with oestrogen receptor-negative tumours. We propose that β3 inhibitors may be more efficacious if used in a neoadjuvant setting, rather than after metastases are established.

We have previously demonstrated that alternagin-C (ALT-C), a disintegrin-like, Cys-rich protein isolated from Bothrops alternatus snake venom, induces human vascular endothelial cell (HUVEC) proliferation and angiogenesis in in vitro and in vivo assays. Therefore this protein could be interesting as a new approach for tissue regeneration studies. However, its primary sequence was not completely determined since the protein isolated from crude venom is usually a mixture of isoforms. Here we describe the transcriptome analysis of B. alternatus from the venom glands of a single male specimen. About 800 good-quality contigs were screened for snake venom metalloproteases/disintegrins, resulting in the following expression profile for these enzymes: 4% for P-I, 7% for P-II and 89% for P-III SVMPs. The PII-SVMP sequence code for RGD-disintegrins and all the expressed PIII-sequences have the ECD adhesive motif. A cDNA sequence coding for an ALT-C homolog was completely sequenced and characterized. Comparative sequence and structural analyses suggested new features that distinguish SVMP classes such as two prolyl endopetidase cleavage sites. All these data add new information on the expression pattern of metalloproteases of B. alternatus venom and may have practical applications for the production of recombinant disintegrins for cell adhesion studies.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, studies investigating the effect of chemotherapy on the metabolism of breast cancer cells are still limited. We used

The connective tissue formed by extracellular matrix (ECM) rich in fibronectin and collagen consists a barrier that cancer cells have to overpass to reach blood vessels and then a metastatic site. Cell adhesion to fibronectin is mediated by αvβ3 and α5β1 integrins through an RGD motif present in this ECM protein, thus making these receptors key targets for cell migration studies. Here we investigated the effect of an RGD disintegrin, DisBa-01, on the migration of human fibroblasts (BJ) and oral squamous cancer cells (OSCC, SCC25) on a fibronectin-rich environment. Time-lapse images were acquired on fibronectin-coated glass-bottomed dishes. Migration speed and directionality analysis indicated that OSCC cells, but not fibroblasts, showed significant decrease in both parameters in the presence of DisBa-01 (1μM and 2μM). Integrin expression levels of the α5, αv and β3 subunits were similar in both cell lines, while β1 subunit is present in lower levels on the cancer cells. Next, we examined whether the effects of DisBa-01 were related to changes in adhesion properties by using paxillin immunostaining and total internal reflection fluorescence TIRF microscopy. OSCCs in the presence of DisBa-01 showed increased adhesion sizes and number of maturing adhesion. The same parameters were analyzed usingβ3-GFP overexpressing cells and showed that β3 overexpression restored cell migration velocity and the number of maturing adhesion that were altered by DisBa-01. Surface plasmon resonance analysis showed that DisBa-01 has 100x higher affinity for αvβ3 integrin than forα5β1 integrin. In conclusion, our results suggest that the αvβ3 integrin is the main receptor involved in cell directionality and its blockage may be an interesting alternative against metastasis.

Adipose tissue development is associated with modifications involving extracellular matrix remodelling, and metalloproteinases play a significant role in this process. Reduced circulating sexual hormones cause impacts on the size, morphology and functions of the adipose tissue, increasing susceptibility to diseases. This study investigated whether exercise training may be an alternative strategy to combat the effects promoted by estrogen decay through modulation in gene expression patterns in the extracellular matrix (ECM) of visceral adipose tissue of ovariectomized rats. Nulliparous rats (n = 40) were randomly distributed into four groups (n = 10/group): sham sedentary (Sh-S), sham resistance training (Sh-Rt), ovariectomized sedentary (Ovx-S) and ovariectomized resistance training (Ovx-Rt). The Sh-S animals did not have any type of training. The body mass and food intake, ECM gene expression, gelatinase MMP-2 activity and adipocyte area were measured. A lack of estrogen promoted an increase in body mass, food intake and the visceral, parametrial and subcutaneous adipocyte areas. The ovariectomy upregulated the expression of MMP-2, MMP-9, TGF-β, CTGF, VEGF-A and MMP-2 activity. On the other hand, resistance training decreased the body mass, food intake and the adipocyte area of the three fat depots analysed; upregulated TIMP-1, VEGF-A and MMP-2 gene expression; downregulated MMP-9, TGF-β and CTGF gene expression; and decreased the MMP-2 activity. We speculate that resistance training on a vertical ladder could play an important role in maintaining and remodelling ECM by modulation in the ECM gene expression and MMP-2 activity, avoiding its destabilization which is impaired by the lack of estrogen.

Progressive fibrosis is a hallmark of the aging heart. Age-related fibrosis is modulated by endurance exercise training; however, little is known concerning the influence of resistance training (RT). Therefore we investigated the chronic effects of high-intensity RT on age-associated alterations of left ventricle (LV) structure, collagen content, matrix metalloproteinase-2 (MMP-2), and extracellular matrix-related gene expression, including transforming growth factor-β (TGF-β). Young adult (3 mo) and aged (21 mo) male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), three times a week for 12 wk. Forty-eight hours posttraining, arterial systolic and diastolic pressure, LV end-diastolic pressure (LVEDP) and dP/d t were recorded. LV morphology, collagen deposition, and gene expression of type I (COL-I) and type III (COL-III) collagen, MMP-2, tissue inhibitor of metalloproteinases-1 (TIMP-1), and TGF-β1 were analyzed by quantitative reverse transcriptase-PCR. MMP-2 content was assessed by zymography. Increased collagen deposition was observed in LV from aged rats. These parameters were modulated by RT and were associated with increased MMP-2 activity and decreased COL-I, TGF-β1, and TIMP-1 mRNA content. Despite the effect of RT on collagen accumulation, there was no improvement on LVEDP and maximal negative LV dP/d t of aged rats. Cardiomyocyte diameter was preserved in all experimental conditions. In conclusion, RT attenuated age-associated collagen accumulation, concomitant to the increase in MMP-2 activity and decreased expression of COL-I, TGF-β1, and TIMP-1 in LV, illustrating a cardioprotective effect of RT on ventricular structure and function. NEW & NOTEWORTHY We demonstrated the beneficial resistance-training effect against age-related left ventricle collagen accumulation in the left ventricle, which was associated with decreased type I collagen (COL-I), transforming growth factor-β1 (TGF-β1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression and matrix metalloproteinase-2 (MMP-2) activity. Our findings suggest for the first time the potential effects of resistance training in modulating collagen accumulation and possibly fibrosis in the aging heart.

Cancer is the second leading cause of death, preceded only by cardiovascular diseases, and there is epidemiological evidence that demonstrate this tendency is emerging worldwide. Brazil has an extensive vegetal biodiversity with more than 55,000 species listed. Such biodiversity collaborates with the finding of compounds which could be the basis for the design of new anti-tumor drugs, with fewer side effects than the conventional chemotherapy used currently. Cedrelone is a limonoid isolated from Trichilia catigua (Meliaceae) which is a native Brazilian plant. This study demonstrates that cedrelone inhibits proliferation, adhesion, migration and invasion of breast tumor cells from the line MDA-MB-231. The effects of cell migration and invasion on MDA-MB-231 cell may be explained, at least in part, by the ability of cedrelone to inhibit MMP activity. We also demonstrate that cedrelone is able to induce apoptosis in MDA-MB-231 cells. There are only a few works investigating the effect of limonoids in cellular processes closely related to tumor progression such as adhesion, migration and invasion. To the best of our knowledge, this is the first work describing the effects of a limonoid on tumor and non-tumor cell adhesion process.

Objective Earlier studies have demonstrated that a disintegrin and metallopeptidase 10 (ADAM10) levels are reduced in Alzheimer's disease (AD) patients compared with healthy subjects. The objective of this study was to evaluate whether platelet ADAM10 levels correlates with the clock drawing test (CDT) scores, which is a simple and a reliable measure of visuospatial ability and executive function in AD patients. Methods Thirty elderly patients with probable AD and 25 healthy patients forming the control group, matched by age, gender, and educational level, were evaluated. Platelet proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ADAM10 was identified by western blotting. The Spearman's correlation coefficient between ADAM10 and CDT was obtained for each group. The areas under the curves were used to compare the receiver operating characteristic curves. Results The CDT scores and platelet ADAM10 expression were significantly different between patients with AD and controls and also along the disease's progression. In AD patients, there was a positive correlation between ADAM10 expression and CDT scores. Among non-AD subjects, no correlation was found. The combination of ADAM10 and CDT was significantly better to confirm the AD diagnosis than the AUCs of ADAM10 and CDT separately. Conclusions The association of blood-based biomarkers, such as ADAM10, and cognitive tests may be helpful for a more reliable AD diagnosis. Copyright © 2013 John Wiley & Sons, Ltd.

Integrin αvβ3 is most likely the foremost modulator of angiogenesis among all known integrins. Recombinant disintegrin DisBa-01, originally obtained from snake venom glands, binds to αvβ3, thereby significantly inhibiting adhesion and generating in vivo anti-metastatic ability. However, its function in mediator production is not clear. Here, we observed that the mediators VEGF-A, IL-8, and TGF-β are not produced by human umbilical vein endothelial cells (HUVEC cell line) or monocyte/macrophage cells (SC cell line) when cells adhered to vitronectin. However, when exposed to DisBa-01, HUVECs produced higher levels of TGF-β, and SC cells produced higher levels of VEGF-A. Nonetheless, HUVECs also showed an enhancement of apoptosis after losing adherence when exposed to disintegrin, which is a characteristic of anoikis. We propose that disintegrin DisBa-01 could be used to modulate integrin αvβ3 functions.

OBJECTIVE: The objective of this study was to assess the effects of resistance training on oxidative stress markers in the livers of ovariectomized rats. METHOD: Adult Sprague-Dawley rats were divided into the following four groups (n = 8 per group): sham-operated sedentary, ovariectomized sedentary, sham-operated resistance training, and ovariectomized resistance training. During the resistance training period, the animals climbed a 1.1-m vertical ladder with weights attached to their tails; the sessions were conducted 3 times per week, with 4-9 climbs and 8-12 dynamic movements per climb. The oxidative stress was assessed by measuring the levels of reduced glutathione and oxidized glutathione, the enzymatic activity of catalase and superoxide dismutase, lipid peroxidation, vitamin E concentrations, and the gene expression of glutathione peroxidase. RESULTS: The results showed significant reductions in the reduced glutathione/oxidized glutathione ratio (4.11±0.65 nmol/g tec), vitamin E concentration (55.36±11.11 nmol/g), and gene expression of glutathione peroxidase (0.49±0.16 arbitrary units) in the livers of ovariectomized rats compared with the livers of unovariectomized animals (5.71±0.71 nmol/g tec, 100.14±10.99 nmol/g, and 1.09±0.54 arbitrary units, respectively). Moreover, resistance training for 10 weeks was not able to reduce the oxidative stress in the livers of ovariectomized rats and induced negative changes in the hepatic anti-oxidative/oxidative balance. CONCLUSION: Our findings indicate that the resistance training program used in this study was not able to attenuate the hepatic oxidative damage caused by ovariectomy and increased the hepatic oxidative stress.

Alzheimer’s disease (AD) is the most common cause of dementia in people above age 65. Platelet studies with ADAM10 have shown that its expression is reduced in AD patients. The aim of this research was to compare the platelet levels of ADAM10 protein in two Brazilian elderly groups, considering the stages of the disease. The SDS-PAGE technique followed by Western blotting was used. Data were analyzed using comparison, correlation and association statistical methods. The results showed reduced platelet ADAM10 levels in AD elderly compared to non-AD subjects. The disease progression intensified this reduction. ADAM10 was the only statistically significant variable (p = 0.01) to increase the AD occurrence probability. The cutoff value of 0.4212 in the receiver operating characteristic curve captured sensitivity and specificity of 70 and 80.77%, respectively. Together with other clinical criteria, ADAM10 seems to be a relevant biomarker tool for early and accurate AD diagnosis.

ADAMs are transmembrane multifunctional proteins that contain disintegrin and metalloprotease domains. ADAMs act in a diverse set of biological processes, including fertilization, inflammatory responses, myogenesis, cell migration, cell proliferation and ectodomain cleavage of membrane proteins. These proteins also have additional functions in pathological processes as cancer and metastasis development. ADAM9 is a member of ADAM protein family that is overexpressed in several types of human carcinomas. The aim of this study was to investigate the role of ADAM9 in hematogenous and lymphatic tumor cell dissemination assisting the development of new therapeutic tools. The role of ADAM9 in the interaction of breast tumor cells (MDA-MB-231) and endothelial cells was studied through RNA silencing. ADAM9 silencing in MDA-MB-231 cells had no influence in expression of several genes related to the metastatic process such as ADAM10, ADAM12, ADAM17, cMYC, MMP9, VEGF-A, VEGF-C, osteopontin and collagen XVII. However, there was a minor decrease in ADAM15 expression but an increase in that of MMP2. Moreover, ADAM9 silencing had no effect in the adhesion of MDA-MB-231 cells to vascular (HMEC-1 and HUVEC) and lymphatic cells (HMVEC-dLyNeo) under flow condition. Nevertheless, siADAM9 in MDA-MB-231 decreased transendothelial cell migration in vitro through HUVEC, HMEC-1 and HMVEC-dLyNeo (50%, 40% and 32% respectively). These results suggest a role for ADAM9 on the extravasation step of the metastatic cascade through both blood and lymph vessels.