RESUMO
The functions of annexin A1 (ANXA1), which is expressed on membranes and in cytoplasmic granules, have been fully described. Nonetheless, the role of this protein in protecting against DNA damage in the nucleus is still emerging and requires further investigation. Here, we investigated the involvement of ANXA1 in the DNA damage response in placental cells. Placenta was collected from ANXA1 knockout mice (AnxA1-/-) and pregnant women with gestational diabetes mellitus (GDM). The placental morphology and ANXA1 expression, which are related to the modulation of cellular response markers in the presence of DNA damage, were analyzed. The total area of AnxA1-/- placenta was smaller due to a reduced labyrinth zone, enhanced DNA damage, and impaired base excision repair (BER) enzymes, which resulted in the induction of apoptosis in the labyrinthine and junctional layers. The placentas of pregnant women with GDM showed reduced expression of AnxA1 in the villous compartment, increased DNA damage, apoptosis, and a reduction of enzymes involved in the BER pathway. Our translational data provide valuable insights into the possible involvement of ANXA1 in the response of placental cells to oxidative DNA damage and represent an advancement in investigations into the mechanisms involved in placental biology.
Assuntos
Anexina A1 , Diabetes Gestacional , Camundongos , Animais , Gravidez , Humanos , Feminino , Placenta/metabolismo , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Anexina A1/metabolismo , Processamento de Proteína Pós-Traducional , Dano ao DNARESUMO
Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.
Assuntos
Anexina A1 , Melanoma , Animais , Camundongos , Anexina A1/metabolismo , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Fagocitose , Microambiente TumoralRESUMO
L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.
Assuntos
Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Saccharomyces , Humanos , Asparaginase/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Antineoplásicos/farmacologiaRESUMO
Maternal neutrophils cells are players in gestational tolerance and fetus delivery. Nonetheless, their actions in each phase of the pregnancy are unknown. We here investigated the role of maternal neutrophil depletion before the blastocyst implantation phase and outcomes in the pregnancy index, placenta, and fetus development. Neutrophils were pharmacologically depleted by i.p. injection of anti-Gr1 (anti-neutrophils; 200 µg) 24 hours after plug visualization in allogeneic-mated C57BL/6/BALB/c mice. Depletion of peripheral neutrophils lasted until 48 hours after anti-Gr1 injection (gestational day 1.5-3.5). On gestational day 5.5, neutrophil depletion impaired the blastocyst implantation, as 50% of pregnant mice presented reduced implantation sites. On gestational day 18.5, neutrophil depletion reduced the pregnancy rate and index, altered the placenta disposition in the uterine horns, and modified the structure of the placenta, detected by reduced junctional zone, associated with decreased numbers of giant trophoblast cells, spongiotrophoblast. Reduced number of placenta cells labeled for vascular endothelial growth factor (VEGF), platelet-endothelial cell adhesion molecule (PECAM-1), and intercellular cell adhesion molecule (ICAM-1), important markers of angiogenesis and adhesiveness, were detected in neutrophil depleted mice. Furthermore, neutrophil depletion promoted a higher frequency of monocytes, natural killers, and T regulatory cells, and lower frequency of cytotoxic T cells in the blood, and abnormal development of offspring. Associated data obtained herein highlight the pivotal role of neutrophils actions in the early stages of pregnancy, and address further investigations on the imbricating signaling evoked by neutrophils in the trophoblastic interaction with uterine epithelium.
Assuntos
Molécula 1 de Adesão Intercelular , Fator A de Crescimento do Endotélio Vascular , Animais , Implantação do Embrião , Feminino , Feto , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Placenta/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
Aim: The low solubility and consequent poor bioavailability of ibuprofen (IBU) is a major drawback that can be overcome by anchoring IBU on ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) as effective multifunctional carriers for drug delivery. Methods: USPIONs were conjugated with glycerol phosphate (USPION-GP) and also co-conjugated with IBU (USPION-GP/IBU), and their in vivo toxicity and anti-inflammatory effects investigated. Phosphate buffer saline (control), IBU, USPION-GP and USPION-GP/IBU were intravenously administered 15 min before lipopolysaccharide-induced peritonitis in male Balb/c mice. Results: 4 h later, USPION bioconjugates did not appear to have caused toxicity to blood leukocytes or caused alterations in the spleen, liver or kidneys. Also, they inhibited lipopolysaccharide-induced neutrophil mobilization into the peritoneum. Conclusion: The absence of systemic toxicity and the unexpected anti-inflammatory action of USPION bioconjugates indicates that they could be a novel and effective approach to administer IBU and warrant further investigation.
Assuntos
Ibuprofeno , Nanopartículas Magnéticas de Óxido de Ferro , Animais , Anti-Inflamatórios/toxicidade , Anti-Inflamatórios não Esteroides/toxicidade , Disponibilidade Biológica , Ibuprofeno/toxicidade , Masculino , Camundongos , SolubilidadeRESUMO
Intravital microscopy (IVM) is an essential experimental approach for evaluating, in real time, cell interactions in the blood and rheological parameters in the microcirculation of the living animals. Different tissues are surgically exposed to the visualization of the microvascular network in optical microscopies connected to video cameras and image software. By evaluating in situ microcirculatory network, IVM allows the visualization and quantification of physiological and pathological processes in the blood or in the adjacent tissues considering the whole system. Therefore, IVM has been used to evaluate the effects and mechanisms of actions in the microvascular network caused by pharmacological or toxic chemical agents. In this chapter, different experimental approaches are described to study the toxic effects and mechanisms of xenobiotics in the microcirculatory network.
Assuntos
Microscopia Intravital/métodos , Microvasos/efeitos dos fármacos , Nanoconjugados/toxicidade , Testes de Toxicidade/métodos , Xenobióticos/toxicidade , Animais , Microscopia Intravital/instrumentação , Microvasos/diagnóstico por imagem , Reologia/métodos , Xenobióticos/farmacocinéticaRESUMO
L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Produtos Biológicos/farmacologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Lisossomos/imunologia , Peptídeo Hidrolases/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Asparaginase/química , Asparaginase/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Galinhas , Relação Dose-Resposta a Droga , Escherichia coli , Feminino , Cavalos , Humanos , Fenômenos Imunogenéticos/fisiologia , Células Jurkat , Lisossomos/química , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/química , Peptídeo Hidrolases/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Estrutura Secundária de ProteínaRESUMO
Embryo implantation into the uterine wall is a highly modulated, complex process. We previously demonstrated that Annexin A1 (AnxA1), which is a protein secreted by epithelial and inflammatory cells in the uterine microenvironment, controls embryo implantation in vivo. Here, we decipher the effects of recombinant AnxA1 in this phenomenon by using human trophoblast cell (BeWo) spheroids and uterine epithelial cells (Ishikawa; IK). AnxA1-treated IK cells demonstrated greater levels of spheroid adherence and upregulation of the tight junction molecules claudin-1 and zona occludens-1, as well as the glycoprotein mucin-1 (Muc-1). The latter effect of AnxA1 was not mediated through IL-6 secreted from IK cells, a known inducer of Muc-1 expression. Rather, these effects of AnxA1 involved activation of the formyl peptide receptors FPR1 and FPR2, as pharmacological blockade of FPR1 or FPR1/FPR2 abrogated such responses. The downstream actions of AnxA1 were mediated through the ERK1/2 phosphorylation pathway and F-actin polymerization in IK cells, as blockade of ERK1/2 phosphorylation reversed AnxA1-induced Muc-1 and claudin-1 expression. Moreover, FPR2 activation by AnxA1 induced vascular endothelial growth factor (VEGF) secretion by IK cells, and the supernatant of AnxA1-treated IK cells evoked angiogenesis in vitro. In conclusion, these data highlight the role of the AnxA1/FPR1/FPR2 pathway in uterine epithelial control of blastocyst implantation.
Assuntos
Anexina A1/metabolismo , Blastocisto/metabolismo , Receptores de Formil Peptídeo/metabolismo , Útero/fisiologia , Actinas/metabolismo , Animais , Linhagem Celular , Claudina-1/metabolismo , Implantação do Embrião , Células Epiteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Mucina-1/metabolismo , Neovascularização Fisiológica , Polimerização , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Agents that inhibit angiogenic factors may prevent the development of hepatocellular carcinoma (HCC). Thus, the objective of this study was to kinetically evaluate the antiangiogenic activity of tributyrin (TB), a butyric acid prodrug, in the promotion stage of hepatocarcinogenesis. For this purpose, the resistant hepatocyte (RH) model was used for induction of preneoplastic lesions in Wistar rats. During the promotion phase, the animals received TB or maltodextrin (MD) as control daily. The rats were killed at three time-points (P1, P2 and P3). Increased expression of Vegfa and Vegfr2 was observed during promotion phase of hepatocarcinogenesis, which was not reversed by TB treatment. However, TB treatment reduced the expression of cluster of differentiation (CD) 34-positive vessels at P3 and α-smooth muscle actin (α-SMA)-positive vessels at P2 compared with MD. Enhanced levels of hypoxia inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinases (pERK) were detected at P3 when compared with P1 and P2 in the MD treatment. TB treatment reduced the levels of HIF-1α and pERK at P3 relative to the MD control. Experiments with human umbilical vein endothelial cells (HUVEC) showed that sodium butyrate (NaBu) inhibited cell migration and tube formation, confirming the antiangiogenic activity of its prodrug TB. In conclusion, antiangiogenic activity of TB is an early event that already occurs in preneoplastic livers, reinforcing its potential chemopreventive effects against HCC.
Assuntos
Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Triglicerídeos/farmacologia , Actinas/genética , Inibidores da Angiogênese/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ácido Butírico/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Polissacarídeos/farmacologia , Pró-Fármacos/farmacologia , Ratos , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Paraquat (PQ) is one of the most widely employed herbicides that is used worldwide and it causes severe toxic effects in humans and animals. A PQ exposition can lead to pulmonary fibrosis (PF) and the mechanisms seem to be linked to oxidative stress, although other pathways have been suggested. Antioxidants can be useful as a therapy, although interventions with this kind of system are still controversial. Hence, this study has investigated the role of ascorbic acid (vitamin C) post-treatment on PQ-induced PF in male C57/BL6 mice. Pulmonary fibrosis was induced by a single PQ injection (10mg/kg; i.p.). The control group received a PQ vehicle. Seven days after the PQ or vehicle injections, the mice received vitamin C (150 mg/kg, ip, once a day) or the vehicle, over the following 7 days. Twenty-four hours after the last dose of vitamin C or the vehicle, the mice were euthanized and their bronchoalveolar lavage fluid (BALF) and their lungs were collected. The data obtained showed that vitamin C reduced the cellular recruitment, the secretion of IL-17 -a cytokine involved in neutrophils migration, TGF-ß-a pro-fibrotic mediator and the collagen deposition. Moreover, vitamin C elevated the superoxide dismutase (SOD) and catalase levels, both antioxidant enzymes, but it did not alter the tracheal contractile response that was evoked by methacholine. Therefore, the researchers have highlighted the mechanisms of vitamin C as being non-invasive and have suggested it as a promising tool to treat lung fibrosis when it is induced by a PQ intoxication.