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Plant phytoprostanes (PhytoPs) are lipid oxidative stress mediators that share structural similarities with mammal prostaglandins (PGs). They have been demonstrated to modulate inflammatory processes mediated by prostaglandins. The present study aims to test the effects of the most abundant oxylipin from Gracilaria longissima, ent-9-D1t-Phytoprostane (9-D1t-PhytoP), on platelet activation and vascular cells as well as clarify possible interactions with platelets and the endothelial EP3 receptor Platelet and monocyte activation was assessed by flow cytometry in the presence of purified 9-D1t-PhytoP. Cell migration was studied using the human Ea.hy926 cell line by performing a scratch wound healing assay. The RNA expression of inflammatory markers was evaluated by RT-PCR under inflammatory conditions. Blind docking consensus was applied to the study of the interactions of selected ligands against the EP3 receptor protein. The 9D1t-PhytoP exerts several pharmacological effects; these include prothrombotic and wound-healing properties. In endothelial cells, 9D1t-PhytP mimics the migration stimulus of PGE2. Computational analysis revealed that 9D1t-PhytP forms a stable complex with the hydrophobic pocket of the EP3 receptor by interaction with the same residues as misoprostol and prostaglandin E2 (PGE2), thus supporting its potential as an EP3 agonist. The potential to form procoagulant platelets and the higher endothelial migration rate of the 9-D1t-PhytoP, together with its capability to interact with PGE2 main target receptor in platelets suggest herein that this oxylipin could be a strong candidate for pharmaceutical research from a multitarget perspective.
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Gracilaria , Animais , Humanos , Receptores de Prostaglandina , Células Endoteliais/metabolismo , Oxilipinas/farmacologia , Ativação Plaquetária , Dinoprostona/metabolismo , Prostaglandinas , Movimento Celular , Receptores de Prostaglandina E Subtipo EP3 , Leucócitos/metabolismo , Mamíferos/metabolismoRESUMO
Food peptides contain a very wide range of diversified structures, which explains their diverse range of functional activities. Proatherogenic endothelium is related to vasoconstriction, inflammation, and oxidative stress. In this line, four synthetic bioactive peptides from dry-cured pork ham, previously identified according to their Angiotensin I Converting Enzyme (ACE) inhibitory capacity and high bioavailability, were tested. Among them, KPVAAP displayed an estimated IC50 of 59.22 µM for human ACE inhibition, and docking simulations demonstrated the consistency of the noncompetitive binding with the protein. The addition of synthetic peptides to human endothelial cells significantly prevents the expression of genes related to endothelial dysfunction and inflammation (eNOS, ICAM-1, VCAM-1, IL-6) and lowers NF-κB activation (all p < 0.05). In silico dockings showed that the four bioactive peptides interact with the regulatory subunit NEMO of the NF-κB transcription factor at the same site as other characterized inhibitors (CC2-LZ region). This is the first study linking experimental and computational approaches that shows NF-κB to be the target of biopeptides of food origin. These multifunctional peptides from dry-cured pork ham make them good candidates for further research into their therapeutic or preventive use to attenuate the inflammatory atherosclerotic process.
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Anti-Inflamatórios/farmacologia , Células Endoteliais/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Proteínas de Carne/química , Simulação de Acoplamento Molecular , Oligopeptídeos/farmacologia , Carne de Porco , Anti-Inflamatórios/química , Sítios de Ligação , Linhagem Celular , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Quinase I-kappa B/química , Oligopeptídeos/química , Ligação ProteicaRESUMO
PURPOSE: Therapeutic exploitation of angiogenesis in breast cancer has been limited by the lack of reliable biomarkers. Circulating small-sized endothelial microparticles (sEMP) are likely to play a significant role as messengers of angiogenesis. Higher levels of EMP have been observed in cancer patients, but their prognostic value in breast cancer is unknown. Our aim was to determine the value of circulating sEMP as a marker of response to chemotherapy in breast cancer. METHODS: We included patients with breast cancer treated with neoadjuvant or first-line chemotherapy. Baseline and post-treatment circulating sEMP (CD144+) were quantified using a flow cytometer approach specifically designed for analysis of small-sized particles (0.1-0.5 µm). Small-sized EMP response was defined as a post-treatment decrease of sEMP larger than the median decrease of sEMP after chemotherapy. Baseline and post-chemotherapy VEGFA levels were determined with ELISA. RESULTS: Forty-four breast cancer patients were included (19 with metastatic and 25 with locally advanced disease). Median levels of sEMP decreased after chemotherapy (P = 0.005). Response to chemotherapy showed a non-significant trend to associate with sEMP response (P = 0.056). A sEMP response was observed in 51% of patients and was associated with better overall survival (HR 0.18; 95% CI 0.04-0.87; P = 0.02) and progression free survival (HR 0.30; 95% CI 0.09-0.99; P = 0.04) in the group of women with metastatic disease. Post-chemotherapy decrease of VEGFA levels was not associated with breast cancer prognosis. CONCLUSIONS: Our results did not support sEMP as a marker of response to chemotherapy. However, our exploratory analysis suggests that in patients with metastatic breast cancer, the decrease of sEMP levels after chemotherapy is associated with better overall and disease free survival and might be superior to VEGFA levels as an angiogenesis-related prognostic marker.
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Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Prognóstico , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/patologia , Intervalo Livre de Doença , Endotélio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Resultado do TratamentoRESUMO
The incidence of thyroid cancer (TC) - the most common endocrine malignancy - has been increasing sharply since the mid-1990s. The rate of TC incidence in both men and women has been faster than any other cancer. Both improved diagnoses (i.e. increased medical surveillance and more sensitive diagnostic tests, such as ultrasound and confirmation via fine-needle aspiration biopsy (FNAB)), and environmental factors detrimental to thyroid health are thought to account for the increased incidence. There are several histological types of thyroid carcinoma including papillary, follicular, medullary, and anaplastic. Determining the type of TC is crucial for prognosis and treatment selection. Unfortunately, approximately 20-30% of patients undergoing FNAB have inconclusive or indeterminate results, leading to unnecessary surgical intervention in 80% of patients with benign nodules. To resolve this diagnostic dilemma, new biomarkers of TC are needed. Proteomic approaches offer an unbiased platform for the comprehensive analysis of the whole proteome. Although mRNA expression is widely considered to be indicative of protein expression, protein levels are the result of protein synthesis and degradation, yet RNA levels are only indicative of protein synthesis. Clinically, there is growing evidence for the role of proteomic and metabolomic technologies in TC biomarker discovery, providing novel information on the molecular events associated with TC, and potentially leading to the identification of novel drug targets. This review thoroughly discusses the importance of novel proteomic and metabolomic approaches to identify new biomarkers for TC.
Assuntos
Biomarcadores Tumorais , Metabolômica/métodos , Proteômica/métodos , Neoplasias da Glândula Tireoide , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Humanos , Espectrometria de Massas , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/metabolismoRESUMO
Multiple factors contribute to the risk of venous thromboembolism (VTE). Platelets have attracted much interest in arterial cardiovascular disease, whereas their role in VTE has received much less attention. Recent evidence suggests that platelets may play a more important role in VTE than previously anticipated. This review discusses the mechanisms that link platelets with venous thrombotic disease and their potential applications as novel risk factors for VTE. In addition, animal studies and randomized clinical trials that highlight the potential effect of antiplatelet therapy in venous thrombosis are evaluated to assess the role of platelets in VTE. The clinical significance of platelets for VTE risk assessment in specific patient cohorts and their role as a suitable therapeutic target for VTE prevention is acknowledged. The role of platelets in VTE is a promising field for future research.
Assuntos
Plaquetas/fisiologia , Medição de Risco/métodos , Transdução de Sinais/fisiologia , Tromboembolia Venosa/fisiopatologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Humanos , Modelos Biológicos , Mutação , Inibidores da Agregação Plaquetária/uso terapêutico , Glicoproteínas da Membrana de Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos , Tromboembolia Venosa/tratamento farmacológico , Tromboembolia Venosa/genéticaRESUMO
This study aimed to examine the mechanisms of cellular activation by small-size platelet microparticles (sPMP) and to present the performance of high-resolution flow cytometry for the analysis of subcellular entities from different origins. Plasma counts of sPMP were analysed in coronary artery disease patients (n = 40) and healthy controls (n = 40). The effect of sPMP and platelet debris (PD) in pathophysiologically relevant doses on platelet and monocyte activation parameters and thrombogenesis was investigated via flow cytometry and thromboelastometry. New generation flow cytometry identifies differences in size, levels and surface molecules of sPMP derived in the absence of stimulus, thrombin activation and platelet disruption. Addition of sPMP resulted in platelet degranulation and P-selectin redistribution to the membrane (P = 0·019) in a dose and time-dependent manner. Blood clotting time decreased after addition of sPMP (P = 0·005), but was not affected by PD. Blocking P-selectin (CD62P) in sPMP markedly reverted the effect on thrombus kinetics (P = 0·035). Exposure to sPMP stimulated monocyte expression of intercellular adhesion molecule-1 (P < 0·03) and decreased monocyte interleukin-6 receptor density (P < 0·01). These results implicate sPMP as a direct source of downstream platelet and monocyte activation. In pathological coronary artery disease conditions, higher levels of sPMP favour a prothrombotic state, partly through P-selectin expression.
Assuntos
Plaquetas/fisiologia , Monócitos/fisiologia , Selectina-P/fisiologia , Ativação Plaquetária/fisiologia , Trombose/sangue , Análise de Variância , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Feminino , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Selectina-P/metabolismo , Tromboelastografia , Trombina/fisiologiaRESUMO
We aimed to provide evidence that blood monocytes belonging to all subsets predominantly circulate in constant and usually reversible interactions with platelets, which are predominantly [Ca2+] dependent. The proportions of monocyte-platelet aggregates (MPAs) attributable to individual monocyte subsets in fresh and promptly processed heparin-anticoagulated blood from 10 healthy subjects (median age 35 years, 50% male) were analysed by flow cytometry and compared to samples anticoagulated with a potent [Ca2+] chelator, ethylenediaminetetraacetic acid (EDTA). Additional experiments with [Ca2+] depletion or supplementation were also performed. Monocytes subsets were defined as CD14++CD16-CCR2+ cells (Mon1), CD14++CD16+CCR2+ cells (Mon2) and CD14+CD16++CCR2- cells (Mon3). Vast majority of monocytes showed aggregation with platelets in heparinised samples, but most monocytes were free of platelets when EDTA was used (p<0.001 for all subsets). Addition of the heparinised blood to EDTA-containing vacutainers reduced the proportion of MPAs to values seen in the directly EDTA-anticoagulated blood (p=0.005 for all subsets). Supplementation with CaCl2 resulted in dose-dependent increase in MPAs (p<0.001 for all subsets). Although the overall trend for the monocyte-platelet interactions was applicable to all monocyte subsets, the proportion of MPAs in heparinised samples was lowest for Mon3 (p<0.0001). In contrast, Mon3 showed the highest proportion of MPAs in EDTA-anticoagulated samples (p=0.004). In healthy subjects monocytes circulate in constant, but predominantly reversible and [Ca2+]-dependent aggregation with platelets. These observations may reflect a complex involvement of platelets in regulation of monocyte activity.
Assuntos
Plaquetas/metabolismo , Cálcio/sangue , Monócitos/metabolismo , Adulto , Plaquetas/citologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Monócitos/citologia , Agregação PlaquetáriaRESUMO
Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.
Assuntos
Proteínas de Transporte , Neoplasias Colorretais , Cinesinas , Proteínas dos Microfilamentos , Invasividade Neoplásica , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte/metabolismo , Cinesinas/metabolismo , Cinesinas/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tionas/farmacologia , Antineoplásicos/farmacologiaRESUMO
In recent years, non-pharmacology treatments and their effectiveness have gained popularity due to their beneficial properties in the prevention of cardiovascular diseases. Phenolic compounds intake provides a natural means of improving in vivo antioxidant status. Thus, the purpose of this review is to discuss the potential benefits of hydroxytyrosol (HT), a phenolic compound with powerful antioxidant and anti-inflammatory properties, in preventing and reducing cardiovascular risk factors, concretely atherosclerosis. Closer inspection of the studies showed a significant improvement of lipid profile, antioxidant capacity and inflammatory state. A note of caution is due in vitro studies because the lack of validated approaches difficult the goodness of fit with the in vivo and clinical research. However, animal and clinical studies were very encouraging, determining HT supplementation useful on inflammation, oxidative stress, endothelial function and cardiovascular diseases in general.
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BACKGROUND: Monocytes are important mediators in the pathophysiology of cardiovascular disease, but only scarce data are available on biological and methodological factors affecting their levels. DESIGN: Three monocyte subsets, CD14(++) CD16(-) CCR2+ (Mon1), CD14(++) CD16(+) CCR2(+) (Mon2), CD14(+) CD16(+) CCR2(-) (Mon3), and monocyte-platelet aggregates (MPAs) were analysed by flow cytometry. The effects of treadmill exercise were assessed on 12 healthy volunteers. Diurnal variation was evaluated in 16 healthy volunteers, and the effects of delayed blood processing were measured in 12 samples. RESULTS: Mon1 were increased when measured 15 min after exercise followed by a reduction at 1 h (P < 0·05 for both). MPAs were significantly reduced at 15 min and 1 h (P < 0·05 for both). There was significant diurnal variation in the numbers of Mon2, which were highest at 6 pm and lowest at 6 am. There were also significant diurnal variations in phagocytic activity of Mon1 and Mon2, which were highest at 12 pm and lowest at 12 am. Monocyte counts remained stable up to 2 h after venipuncture. MPAs were significantly increased at 2 h and increased further by 4 h after sampling. CONCLUSIONS: Monocyte subset Mon2 and monocyte phagocytic activity undergo significant diurnal variation. A single bout of exercise causes a temporal increase in monocytes and a reduction in MPAs. Monocyte subset counts should be analysed within 2 h of blood sampling, whereas measurement of MPAs and monocyte CD14 and CD16 expression should be performed within 1 h.
Assuntos
Plaquetas/metabolismo , Ritmo Circadiano , Exercício Físico/fisiologia , Monócitos/metabolismo , Agregação Plaquetária/fisiologia , Adulto , Teste de Esforço/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , MasculinoRESUMO
Organophosphorus insecticides and nerve agents irreversibly inhibit serine hydrolase superfamily enzymes. One enzyme of this superfamily, the industrially important (for ß-lactam antibiotic synthesis) AXE/CAH (acetyl xylan esterase/cephalosporin acetyl hydrolase) from the biotechnologically valuable organism Bacillus pumilus, exhibits low sensitivity to the organophosphate paraoxon (diethyl-p-nitrophenyl phosphate, also called paraoxon-ethyl), reflected in a high K(i) for it (~5 mM) and in a slow formation (t(½)~1 min) of the covalent adduct of the enzyme and for DEP (E-DEP, enzyme-diethyl phosphate, i.e. enzyme-paraoxon). The crystal structure of the E-DEP complex determined at 2.7 Å resolution (1 Å=0.1 nm) reveals strain in the active Ser¹8¹-bound organophosphate as a likely cause for the limited paraoxon sensitivity. The strain results from active-site-size limitation imposed by bulky conserved aromatic residues that may exclude as substrates esters having acyl groups larger than acetate. Interestingly, in the doughnut-like homohexamer of the enzyme, the six active sites are confined within a central chamber formed between two 60°-staggered trimers. The exclusive access to this chamber through a hole around the three-fold axis possibly limits the size of the xylan natural substrates. The enzyme provides a rigid scaffold for catalysis, as reflected in the lack of movement associated with paraoxon adduct formation, as revealed by comparing this adduct structure with that also determined in the present study at 1.9 Å resolution for the paraoxon-free enzyme.
Assuntos
Acetilesterase/química , Bacillus/enzimologia , Proteínas de Bactérias/química , Resistência Microbiana a Medicamentos , Organofosfatos/metabolismo , Paraoxon/química , Paraoxon/metabolismo , Acetilesterase/antagonistas & inibidores , Acetilesterase/metabolismo , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Cefalosporinas/química , Cefalosporinas/metabolismo , Cristalografia por Raios X , Paraoxon/farmacologia , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologiaRESUMO
We thank Dr. López-Moreno for the comment [...].
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Doenças Cardiovasculares , Carne de Porco , Carne Vermelha , Animais , Suínos , Humanos , Pressão Sanguínea , Doenças Cardiovasculares/prevenção & controle , Fatores de Risco , Nutrientes , Biomarcadores , Fatores de Risco de Doenças CardíacasRESUMO
The good clinical results of immune checkpoint inhibitors (ICIs) in recent cancer therapy and the success of RNA vaccines against SARS-nCoV2 have provided important lessons to the scientific community. On the one hand, the efficacy of ICI depends on the number and immunogenicity of tumor neoantigens (TNAs) which unfortunately are not abundantly expressed in many cancer subtypes. On the other hand, novel RNA vaccines have significantly improved both the stability and immunogenicity of mRNA and its efficient delivery, this way overcoming past technique limitations and also allowing a quick vaccine development at the same time. These two facts together have triggered a resurgence of therapeutic cancer vaccines which can be designed to include individual TNAs and be synthesized in a timeframe short enough to be suitable for the tailored treatment of a given cancer patient.In this chapter, we explain the pipeline for the synthesis of TNA-carrying RNA vaccines which encompasses several steps such as individual tumor next-generation sequencing (NGS), selection of immunogenic TNAs, nucleic acid synthesis, drug delivery systems, and immunogenicity assessment, all of each step comprising different alternatives and variations which will be discussed.
Assuntos
Vacinas Anticâncer , Neoplasias , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Vacinas Sintéticas , Vacinas de mRNARESUMO
BACKGROUND: Evidence suggests that bioactive peptides reduce hypertension and affect certain metabolic pathways. METHODS: Fifty-four volunteers with stage 1 prehypertension and/or hypercholesterolemia and/or basal glucose >100 mg/dL were recruited and randomized to pork dry-cured ham (n = 35) or cooked ham (placebo group; n = 19) for 28 days. After a wash-out period, meat products were changed for 28 additional days. Bioactive peptides composition and enzyme inhibitory activities of both products were characterized. Treatment comparisons for the main effects were made using a two (treatment) × two (times) repeated measures minus the effect of cooked ham (placebo). RESULTS: 24 h mean systolic and diastolic pressures decreased up to 2.4 mmHg in the dry-cured ham period (treatment effect, p = 0.0382 y p = 0.0233, respectively) as well as the number of systolic pressure measures > 135 mmHg (treatment effect, p = 0.0070). Total cholesterol levels also decreased significantly after dry-cured ham intake (p = 0.049). No significant differences were observed between the two treatments for basal glucose, HOMA-IR index and insulin levels (p > 0.05). However, a significant rise of ghrelin levels was observed (treatment effect, p = 0.0350), while leptin plasma values slightly decreased (treatment effect, p = 0.0628). CONCLUSIONS: This study suggested the beneficial effects of regular dry-cured ham consumption on the improvement of systolic/diastolic blood pressures and facilitated the maintenance of metabolic pathways, which may be beneficial in the primary prevention of cardiovascular disease.
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Pressão Sanguínea , Dieta/métodos , Hipercolesterolemia/dietoterapia , Carne de Porco , Pré-Hipertensão/dietoterapia , Adulto , Idoso , Animais , Biomarcadores/análise , Fatores de Risco Cardiometabólico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/prevenção & controle , Estudos Cross-Over , Ingestão de Alimentos/fisiologia , Feminino , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Pré-Hipertensão/complicações , Pré-Hipertensão/fisiopatologia , Suínos , Adulto JovemRESUMO
The aim of this study was to obtain solid carvacrol-cyclodextrin (CD) complexes for use in the pharmaceutical industry. To this end, the complexation of carvacrol at different pH values was studied in detail, to determine the type of CD and the reaction environment that supported the highest amount of encapsulated carvacrol. Evidence of the capability of hydroxypropyl-ß-cyclodextrins (HP-ß-CD) to form inclusion complexes with carvacrol (KC = 5042 ± 176 L mol-1) and more high complexation efficiency (2.824) was demonstrated for HP-ß-CDs using two different energy sources, ultrasound (US) (KC = 8129 ± 194 L mol-1 24 h) and microwave irradiation (MWI) (KC = 6909 ± 161 L mol-1), followed by spraying the resulting solution in a spray dryer. To confirm complex formation, the complexes were characterized using various instrumental methods to corroborate the carvacrol incorporation into the hydrophobic cavity of HP-ß-CD. The obtained carvacrol solid complexes were analyzed by 1H nuclear magnetic resonance (1H-NMR) and 2D nuclear magnetic resonance (ROSEY), differential scanning calorimetry (DSC), thermogravimetric analysis (TG) and Fourier transform infrared spectroscopy (FTIR) characterization. The structures of the resulting complexes were also characterized by molecular modeling. Furthermore, 1 mM HP-ß-CD-carvacrol complex has been shown to reduce cell proliferation in HCT-116 colorectal cancer cells by 43%, much more than in a healthy lung fibroblast MRC-5 cell line (11%).
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Circulating microparticles released from various cell types are present in healthy individuals and the number and composition of their membrane vary in different disorders. Long considered to be cellular debris, microparticles have been recently identified as regulatory vectors of intercellular cross-talk. Indeed, circulating microparticles represent a heterogeneous mixture of spheroids of diverse surface membrane glycoproteins and lipids, with diverse cytoplasm components, the pattern of which depends on the type of stimulation and pathophysiology of parental cells. Despite extensive research into the procoagulant and proinflammatory properties of microparticles, there are few data that can provide information on the mechanism(s) of their formation and biological effects. Although several mechanisms of microparticle release have been suggested, the precise order of the events associated with key features of microparticle formation, transmembrane phosphatidylserine redistribution and cytoskeleton disruption remain to be clarified. In this review, we provide an overview of the molecular mechanisms involved in microparticle formation, as well as the diverse physiological and pathological roles they are able to undertake. Understanding the mechanism(s) governing microparticle release processes may be critical to understanding their precise role in various pathophysiological processes and thus indicate new potential routes to therapy.
Assuntos
Comunicação Celular , Micropartículas Derivadas de Células , Receptor Cross-Talk , HumanosRESUMO
BACKGROUND: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. METHODS: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. RESULTS: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. CONCLUSION: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner.
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Tumor invasion and metastasis involve processes in which actin cytoskeleton rearrangement induced by Fascin1 plays a crucial role. Indeed, Fascin1 has been found overexpressed in tumors with worse prognosis. Migrastatin and its analogues target Fascin1 and inhibit its activity. However, there is need for novel and smaller Fascin1 inhibitors. The aim of this study was to assess the effect of compound G2 in colorectal cancer cell lines and compare it to migrastatin in in vitro and in vivo assays. Molecular modeling, actin-bundling, cell viability, inmunofluorescence, migration, and invasion assays were carried out in order to test anti-migratory and anti-invasive properties of compound G2. In addition, the in vivo effect of compound G2 was evaluated in a zebrafish model of invasion. HCT-116 cells exhibited the highest Fascin1 expression from eight tested colorectal cancer cell lines. Compound G2 showed important inhibitory effects on actin bundling, filopodia formation, migration, and invasion in different cell lines. Moreover, compound G2 treatment resulted in significant reduction of invasion of DLD-1 overexpressing Fascin1 and HCT-116 in zebrafish larvae xenografts; this effect being less evident in Fascin1 known-down HCT-116 cells. This study proves, for the first time, the in vitro and in vivo anti-tumoral activity of compound G2 on colorectal cancer cells and guides to design improved compound G2-based Fascin1 inhibitors. KEY MESSAGES: ⢠Fascin is crucial for tumor invasion and metastasis and is overexpressed in bad prognostic tumors. ⢠Several adverse tumors overexpress Fascin1 and lack targeted therapy. ⢠Anti-fascin G2 is for the first time evaluated in colorectal carcinoma and compared with migrastatin. ⢠Filopodia formation, migration activity, and invasion in vitro and in vivo assays were performed. ⢠G2 blocks actin structures, migration, and invasion of colorectal cancer cells as fascin-dependent.
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Antineoplásicos/uso terapêutico , Proteínas de Transporte/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Indazóis/uso terapêutico , Proteínas dos Microfilamentos/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Embrião não Mamífero , Humanos , Indazóis/farmacologia , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Invasividade Neoplásica , Peixe-ZebraRESUMO
Serrated adenocarcinoma (SAC) is more invasive, has worse outcomes than conventional colorectal carcinoma (CRC), and is characterized by frequent resistance to anti-epidermal growth factor receptor (EGFR) and overexpression of fascin1, a key protein in actin bundling that plays a causative role in tumor invasion and is overexpressed in different cancer types with poor prognosis. In silico screening of 9591 compounds, including 2037 approved by the Food and Drug Administration (FDA), was performed, and selected compounds were analyzed for their fascin1 binding affinity by differential scanning fluorescence. The results were compared with migrastatin as a typical fascin1 inhibitor. In silico screening and differential scanning fluorescence yielded the FDA-approved antidepressant imipramine as the most evident potential fascin1 blocker. Biophysical and different in vitro actin-bundling assays confirm this activity. Subsequent assays investigating lamellipodia formation and migration and invasion of colorectal cancer cells in vitro using 3D human tissue demonstrated anti-fascin1 and anti-invasive activities of imipramine. Furthermore, expression profiling suggests the activity of imipramine on the actin cytoskeleton. Moreover, in vivo studies using a zebrafish invasion model showed that imipramine is tolerated, its anti-invasive and antimetastatic activities are dose-dependent, and it is associated with both constitutive and induced fascin1 expression. This is the first study that demonstrates an antitumoral role of imipramine as a fascin1 inhibitor and constitutes a foundation for a molecular targeted therapy for SAC and other fascin1-overexpressing tumors.
Assuntos
Antidepressivos/farmacologia , Proteínas de Transporte/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Imipramina/farmacologia , Proteínas dos Microfilamentos/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , Macrolídeos/farmacologia , Invasividade Neoplásica/patologia , Piperidonas/farmacologia , Peixe-ZebraRESUMO
The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65 degrees C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new alpha/beta hydrolase family different from IV and VI.