Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.753
Filtrar
1.
BMC Cancer ; 21(1): 438, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33879127

RESUMO

BACKGROUND: Daunorubicin is used clinically in the treatment of myeloma, acute lymphatic and myelocytic leukaemia. The toxic lesions caused by daunorubicin induce various modes of cell death, including apoptosis. Apoptosis is highly regulated programmed cell death that can be initiated mainly via two pathways, through death receptors (extrinsic) or involvement of the mitochondria (intrinsic). Induction of apoptosis via these pathways has been alluded following treatment with daunorubicin, but never compared in acute lymphoblastic leukaemia over a time course. METHODS: This study investigated the mechanisms of daunorubicin induced apoptosis in the treatment of CCRF-CEM, MOLT-4 (acute T-lymphoblastic leukaemia) and SUP-B15 (acute B-lymphoblastic leukaemia) cells. Cells were treated with daunorubicin for 4 h, and then placed in recovery medium (without daunorubicin) for 4 h, 12 h and 24 h. Apoptotic response was analysing using annexin-V expression, caspase activity, mitochondrial membrane potential change and an array to detect 43 apoptotic proteins. RESULTS: Daunorubicin induced apoptosis in all leukemic cell lines, but with different levels and duration of response. Both apoptosis levels and caspase activity increased after four hours recovery then declined in CCRF-CEM and MOLT-4 cells. However, SUP-B15 cells displayed initially comparable levels but remained elevated over the 24 h assessment period. Changes in mitochondrial membrane potential occurred in both MOLT-4 and CCRF-CEM cells but not in SUP-B15 cells. Expression of apoptotic proteins, including Bcl-2, Bax, caspase 3 and FADD, indicated that daunorubicin potentially induced both extrinsic and intrinsic apoptosis in both CCRF-CEM and MOLT-4 cells, but only extrinsic apoptosis in SUP-B15 cells. CONCLUSIONS: This study describes variations in sensitivities and timing of apoptotic responses in different leukaemia cell lines. These differences could be attributed to the lack of functional p53 in coordinating the cells response following cytotoxic treatment with daunorubicin, which appears to delay apoptosis and utilises alternative signalling mechanisms that need to be further explored.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Daunorrubicina/farmacologia , Anexina A5/genética , Anexina A5/metabolismo , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Daunorrubicina/uso terapêutico , Humanos , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos
2.
Mol Med Rep ; 23(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33760105

RESUMO

The present study aimed to determine the anticancer effect of the herbal mixture extract C5E in the pancreatic cancer cell line, PANC­1, in the absence or presence of gemcitabine treatment, a chemotherapeutic drug used for the treatment of pancreatic cancer. The anticancer effects of C5E, gemcitabine and C5E plus gemcitabine in PANC­1 cells following 72 h of treatment were investigated. The effect of each treatment on cell cycle arrest, apoptosis and the proportion of side population (SP) cells was determined using flow cytometric analysis following propidium iodide (PI), Annexin V­FITC/PI double staining and Hoechst 33342 staining, respectively. SP cells share similar characteristics to cancer stem­like cells, and a reduction in the SP is considered to be indicative of an anticancer effect. The percentage of SP cells and the cell viability of general PANC­1 cells were significantly decreased in response to all treatments. The percentage of SP cells was reduced from 8.2% (control) to 3.9, 7.2 and 5.1% following the treatment with C5E, gemcitabine and the co­treatment, respectively. All three treatments were discovered to inhibit cell viability by arresting the cell cycle at the S phase and promoted cell death by inducing early apoptosis, with the levels of apoptosis being increased from 1.9% (control) to 7.3, 2.5 and 12.0% following the treatment with C5E, gemcitabine and the co­treatment, respectively. The mRNA expression levels of sonic hedgehog, which is implicated in the development of certain types of cancer, were downregulated to a greater extent following the co­treatment with C5E and gemcitabine compared with the treatment with either C5E or gemcitabine alone. As the co­treatment with gemcitabine and C5E was more effective than each individual treatment, the present study suggested that the combined treatment may exhibit synergistic effects in PANC­1 cells.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Extratos Vegetais/farmacologia , Anexina A5/genética , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Fluoresceína-5-Isotiocianato/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Medicina Herbária , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Extratos Vegetais/química
3.
Methods Mol Biol ; 2279: 213-223, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33683697

RESUMO

Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.


Assuntos
Anexina A5 , Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Fluoresceína-5-Isotiocianato/análogos & derivados , Neoplasias Pulmonares , Propídio/química , Células A549 , Anexina A5/química , Anexina A5/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia
4.
Methods Mol Biol ; 2240: 243-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33423238

RESUMO

Chemical compounds induce cytotoxicity by various mechanisms, including interference in membrane integrity, metabolism, cellular component degradation or release, and cell division. Between the classic death pathways, namely, autophagy, apoptosis, and necrosis, apoptosis have been in the focus for the last several years as an important pathway for the toxicity of different types of xenobiotics. Because of that, having the tools to evaluate it is key for understanding and explaining the toxicodynamics of different classes of substances. Here, we describe a wide array of classic assays that can be easily implemented to evaluate apoptosis induction.


Assuntos
Apoptose/efeitos dos fármacos , Bioensaio , Mitocôndrias/efeitos dos fármacos , Testes de Toxicidade , Animais , Anexina A5/metabolismo , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo
5.
Phys Chem Chem Phys ; 23(5): 3519-3530, 2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33514968

RESUMO

Glycosaminoglycans (GAGs) are anionic, periodic, linear polysaccharides which are composed of periodic disaccharide units. They play a vital role in many biological processes ongoing in the extracellular matrix. In terms of computational approaches, GAGs are very challenging molecules due to their high flexibility, periodicity, predominantly electrostatic-driven nature of interactions with their protein counterparts and potential multipose binding. Furthermore, the molecular mechanisms underlying GAG-mediated interactions are not fully known yet, and experimental techniques alone are not always sufficient to gain insights into them. The aim of this study was to characterize protein-ion-GAG complexes for the systems where ions are directly involved in GAG binding. Molecular docking, molecular dynamics and free energy calculation approaches were applied to model and rigorously analyse the interactions between annexins (II and V), calcium ions (Ca2+) and heparin (HP). The computational data were examined and discussed in the context of the structural data previously reported by the crystallographic studies. The computational results confirm that the presence of Ca2+ has a tremendous impact on the annexin-HP binding site. This study provides a general computational pipeline to discover the complexity of protein-GAG interactions and helps to understand the role of ions involved at the atomic level. The limitations of the applied protocols are described and discussed pointing at the challenges persisting in the state-of-the-art in silico tools to study protein-ion-GAG systems.


Assuntos
Anexina A2/metabolismo , Anexina A5/metabolismo , Cálcio/metabolismo , Heparina/metabolismo , Animais , Anexina A2/química , Anexina A5/química , Cálcio/química , Heparina/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Ratos , Termodinâmica
6.
Methods Mol Biol ; 2241: 113-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486732

RESUMO

Eosinophil apoptosis (programmed cell death) plays an important role in several inflammatory and allergic conditions. Apoptosis triggers various mechanisms including activation of cysteine-aspartic proteases (caspases) and is characterized by morphological and biochemical changes. These include cellular condensation, nuclear fragmentation, increased mitochondrial permeability with loss of membrane potential, and exposure of phosphatidylserine on the cell membrane. A greater understanding of apoptotic mechanisms, subsequent phagocytosis (efferocytosis), and regulation of these processes is critical to understanding disease pathogenesis and development of potential novel therapeutic agents. Release of soluble factors and alterations to surface marker expression by eosinophils undergoing apoptosis aid them in signaling their presence to the immediate environment, and their subsequent recognition by phagocytic cells such as macrophages. Uptake of apoptotic cells usually suppresses inflammation by restricting proinflammatory responses and promoting anti-inflammatory and tissue repair responses. This, in turn, promotes resolution of inflammation. Defects in the apoptotic or efferocytosis mechanisms perpetuate inflammation, resulting in chronic inflammation and enhanced disease severity. This can be due to increased eosinophil life span or cell necrosis characterized by loss of cell membrane integrity and release of toxic intracellular mediators. In this chapter, we detail some of the key assays that are used to assess eosinophil apoptosis, as well as the intracellular signaling pathways involved and phagocytic clearance of these cells.


Assuntos
Apoptose/fisiologia , Eosinófilos/citologia , Imuno-Histoquímica/métodos , Fagocitose/fisiologia , Anexina A5/química , Apoptose/imunologia , Transporte Biológico , Caspases/metabolismo , Eosinófilos/fisiologia , Humanos , Inflamação/metabolismo , Macrófagos/metabolismo , Potenciais da Membrana/fisiologia , Microscopia/métodos , Microscopia Eletrônica/métodos , Mitocôndrias/metabolismo , Fagócitos/metabolismo , Fagócitos/fisiologia , Fagocitose/imunologia , Propídio/química , Transdução de Sinais
7.
Sci Rep ; 10(1): 21821, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311633

RESUMO

Cancer cells are able to reach distant tissues by migration and invasion processes. Enhanced ability to cope with physical stresses leading to cell membrane damages may offer to cancer cells high survival rate during metastasis. Consequently, down-regulation of the membrane repair machinery may lead to metastasis inhibition. We show that migration of MDA-MB-231 cells on collagen I fibrils induces disruptions of plasma membrane and pullout of membrane fragments in the wake of cells. These cells are able to reseal membrane damages thanks to annexins (Anx) that are highly expressed in invasive cancer cells. In vitro membrane repair assays reveal that MDA-MB-231 cells respond heterogeneously to membrane injury and some of them possess a very efficient repair machinery. Finally, we show that silencing of AnxA5 and AnxA6 leads to the death of migrating MDA-MB-231 cells due to major defect of the membrane repair machinery. Disturbance of the membrane repair process may therefore provide a new avenue for inhibiting cancer metastasis.


Assuntos
Anexina A5/metabolismo , Membrana Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Membrana Celular/patologia , Sobrevivência Celular , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia
8.
Molecules ; 25(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371436

RESUMO

In the present study, the synthesis of gold nanoparticles (AuNPs) loaded with methotrexate (MTX) has been carried out in order to obtain controlled size and monodispersed nanocarriers of around 20 nm. The characterization study shows metallic AuNPs with MTX polydispersed on the surface. MTX is linked by the replacement of citrate by the MTX carboxyl group. The drug release profiles show faster MTX release when it is conjugated, which leads to the best control of plasma concentration. Moreover, the enhanced release observed at pH 5 could take advantage of the pH gradients that exist in tumor microenvironments to achieve high local drug concentrations. AuNP-MTX conjugates were tested by flow cytometry against lung (A-549) and colon (HTC-116) cancer cell lines. Results for A-549 showed a weaker dose-response effect than for colon cancer ones. This could be related to the presence of folate receptors in line HTC-116 in comparison to line A-549, supporting the specific uptake of folate-conjugated AuNP-MTX by folate receptor positive tumor cells. Conjugates exhibited considerably higher cytotoxic effects compared with the effects of equal doses of free MTX. Annexin V-PI tests sustained the cell death mechanism of apoptosis, which is normally disabled in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Ouro/química , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas Metálicas/química , Metotrexato/farmacologia , Células A549 , Anexina A5/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias do Colo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos/fisiologia , Ácido Fólico/farmacologia , Células HCT116 , Humanos , Concentração de Íons de Hidrogênio , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Metotrexato/química , Microambiente Tumoral/efeitos dos fármacos
9.
Int J Nanomedicine ; 15: 4441-4452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606688

RESUMO

Purpose: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated. Methods: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment. Results: NPs showed to be spherical in shape with an average size of 45-55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 µg/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis. Conclusion: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems.


Assuntos
Dano ao DNA , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos
10.
Am J Respir Cell Mol Biol ; 63(4): 519-530, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32628869

RESUMO

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Pulmão/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Anexina A5/metabolismo , Movimento Celular/efeitos dos fármacos , Desoxiglucose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicólise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Pulmão/metabolismo , Masculino , Fosforilação Oxidativa/efeitos dos fármacos , Propídio/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
11.
Anticancer Res ; 40(5): 2739-2749, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366419

RESUMO

BACKGROUND/AIM: Ciprofloxacin has been used as an antibiotic in the clinic for decades. Recently, ciprofloxacin and its derivatives have shown promising anti-proliferative and cytotoxic activities against several malignant cells. The aim of this study was to investigate the effect of a new derivative of ciprofloxacin on colorectal cancer (HCT116) and non-small lung carcinoma (A549) cells. MATERIALS AND METHODS: Cell viability was detected by the MTT assay. Flow cytometry was used to examine the cell cycle and apoptosis. Expression of bax, bcl2, p53 and p21 was investigated by qRT-PCR and western blotting. RESULTS: Ciprofloxacin-derivative had an anti-proliferative effect on both cell lines in a concentration-dependent manner and caused cell cycle arrest at the G2/M phase and apoptosis. p53 and Bax proteins were overexpressed, while p21 and bcl2 gene expression was decreased after treatment with the ciprofloxacin derivative. CONCLUSION: This new ciprofloxacin derivative can be potentially used for the treatment of colorectal cancer and non-small lung carcinoma.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciprofloxacino/farmacologia , Células A549 , Anexina A5/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Concentração Inibidora 50 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
12.
Adv Clin Exp Med ; 29(5): 603-609, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32469166

RESUMO

BACKGROUND: Treatment with cyclosporine A (CsA), a calcineurin inhibitor, is effective in children with difficult idiopathic nephrotic syndrome (INS). Prolonged CsA treatment can result in several adverse effects, the most significant being nephrotoxicity (CsAN). The plasma and urine levels of the proteins annexin V (AnV) and uromodulin (UM) were investigated in order to assess their usefulness as indicators of early-stage CsAN. Uromodulin is considered a distal tubular damage marker. Annnexin V is present in the distal tubules. OBJECTIVES: To measure AnV in children with INS receiving CsA treatment and to assess the usefulness of this biomarker for monitoring CsAN and as an indicator of changes in the distal tubules of the nephron. MATERIAL AND METHODS: The prospective study included 30 patients with INS and 22 controls. Plasma and urinary AnV levels were measured 3 times: before CsA treatment, and after 6 and 12 months of therapy. The AnV levels were compared to those of UM. RESULTS: The urinary AnV and UM levels were significantly higher in the INS patients before CsA therapy in comparison to the reference group. A progressive increase of urinary AnV was observed after 6 and 12 months of therapy. Urinary UM only increased after 6 months. No significant correlations were found between plasma and urinary concentrations of the proteins studied. CONCLUSIONS: The increased urinary excretion of AnV in children with INS receiving CsA treatment may suggest its usefulness as an early marker of subclinical CsAN. Annexin V seems to be a more sensitive indicator of tubular damage in the course of CsA therapy than UM, though large, multicenter studies are needed.


Assuntos
Anexina A5/sangue , Anexina A5/urina , Inibidores de Calcineurina/uso terapêutico , Ciclosporina/uso terapêutico , Imunossupressores/uso terapêutico , Síndrome Nefrótica/tratamento farmacológico , Estudos de Casos e Controles , Criança , Feminino , Humanos , Masculino , Síndrome Nefrótica/sangue , Síndrome Nefrótica/urina , Estudos Prospectivos , Resultado do Tratamento , Uromodulina/sangue , Uromodulina/urina
13.
Medicine (Baltimore) ; 99(17): e19848, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32332640

RESUMO

Xiakemycin A (XKA), a new antibiotic in the pyranonaphthoquinone family, shows antitumor activity. However, the type of cell death induced by XKA remains elusive. In this study, we aim to investigate the type of death induced by XKA in hepatic cancer.The apoptotic features, such as chromatic agglutination, reactive oxygen species generation and membrane potential of mitochondria, in HepG2 cells treated by XKA were measured by Hoechst 33342 staining and flow cytometry. Apoptosis of HepG2 cells treated with XKA was determined by Annexin V-FITC/propidium iodide double staining and Western blot analysis, respectively.XKA had a significant dose-dependent elevation of chromatic agglutination, reactive oxygen species generation, Annexin V and propidium iodide staining, decrease of membrane potential. Meanwhile, in apoptotic HepG2 cells induced by XKA, robust increment was noticed in p53 expression, cleavage of PARP, caspase-3, and caspase-9.XKA showed potent inhibitory effects on the proliferation of HepG2 cells. Such phenomenon may be related to activation of the apoptotic pathway.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Naftoquinonas/farmacologia , Anexina A5/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Caspase 3/metabolismo , Caspase 9/metabolismo , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Propídio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
14.
Cell Physiol Biochem ; 54(2): 287-302, 2020 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-32246616

RESUMO

BACKGROUND/AIMS: Galectin 3 (GAL-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. GAL-3 has been associated with heart failure and was linked to increased risk of death in a number of studies. GAL-3 was found to be up regulated in animal models of heart failure as well as myocardial infarction (MI). The objective of his study is to test if high GAL-3 after myocardial infarction has a protective role on the heart through its anti-apoptotic and anti-necrotic functions. METHODS: Male C57B6/J mice and GAL-3 knockout (KO) mice were used for permanent ligation of the left anterior descending artery of the heart to create infarction in the anterior myocardium. Heart and plasma samples were collected 24 hours after the induction of MI and were used for immunohistochemistry, Tunnel procedure, electron microscopy and enzyme linked immunosorbent assay (ELISA). RESULTS: Our results show that the significant increase in GAL-3 levels in the left ventricle at 24-hour following MI is associated with significant lower levels of pro-apoptotic proteins; cytochrome c, Bax, annexin V, cleaved caspase-3 and a higher levels of anti-apoptotic protein Bcl2 in GAL-3 wild MI group than GAL-3 KO group. We also have identified the anti-apoptotic activity of GAL-3 is mediated through a significant increase in Akt-1, NF kappa-B and beta- catenin proteins. In addition, we have identified the antiapoptotic activity is mediated through a significant lower levels of cathepsin-D protein. CONCLUSION: We conclude that the increased levels of GAL-3 at 24-hour following MI regulate antiapoptotic mechanisms in the myocardium that will shape the future course of the disease. We also identified that the anti-apoptotic mechanisms are likely mediated through interaction of GAL-3 with Akt-1, NF kappa-B, beta- catenin and cathepsin D proteins.


Assuntos
Apoptose/genética , Caspase 3/metabolismo , Galectina 3/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Anexina A5/metabolismo , Catepsinas/metabolismo , Citocromos c/metabolismo , Modelos Animais de Doenças , Galectina 3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/ultraestrutura , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , beta Catenina/metabolismo
15.
Sci Rep ; 10(1): 4057, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132597

RESUMO

The immature preterm kidney is likely to be vulnerable to acute kidney injury (AKI). However, the biomarkers currently used for AKI are not sensitive or specific and are also inadequate for the timely detection of AKI in preterm infants. The objectives of this study were to identify novel urinary biomarkers of AKI using proteomic techniques, and to verify and validate that the candidates can serve as early predictive biomarkers for AKI. In total, 1,810 proteins were identified in the discovery phase. Among those proteins, 174 were selected as the 1st targeted proteins. A total of 168 proteins were quantified, and the levels of 6 were significantly increased in the AKI group in the verification phase. Using a clinical assay, the results were confirmed and validated using samples of the first urine after birth from the biorepository. Finally, enzyme-linked immunosorbent assays revealed that the levels of annexin A5, neutrophil gelatinase-associated lipocalin (NGAL), and protein S100-P were significantly higher in the samples of the first urine from patients with AKI than in those from patients without AKI. In conclusion, urinary annexin A5, NGAL and protein S100-P levels are promising biomarkers for early, accurate prediction of AKI in preterm infants.


Assuntos
Lesão Renal Aguda/urina , Anexina A5/urina , Doenças do Recém-Nascido/urina , Recém-Nascido Prematuro/urina , Lipocalina-2/urina , Proteômica , Proteínas S100/urina , Biomarcadores/urina , Feminino , Humanos , Lactente , Recém-Nascido , Masculino
16.
Biochim Biophys Acta Biomembr ; 1862(6): 183231, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119860

RESUMO

Aging of red blood cells (RBCs) is associated with alteration in a wide range of RBC features, occurring each on its own timescale. A number of these changes are interrelated and initiate a cascade of biochemical and structural transformations, including band-3 clustering and phosphatidylserine (PS) externalization. Using specific band-3 clustering agents (acridine orange (AO) and ZnCl2), we examined whether treatment of RBCs with these agents may affects PS externalization and whether this process is Ca2+-dependent. RBCs were isolated from the blood of eight healthy donors upon obtaining their informed consent. The suspension was supplemented with increasing concentrations of AO or ZnCl2 (from 0.5 to 2.0 mM) and incubated at 25 °C for 60 min. To detect PS at the RBC surface, we used allophycocyanin-conjugated recombinant human Annexin V. We demonstrated, that treatment of RBCs with both clustering agents caused an elevation in the percent of cells positively labeled by Annexin-V (RBCPS), and that this value was not dependent on the presence of calcium in the buffer: RBCs treated with AO in the presence of either EDTA, EGTA or calcium exhibited similar percentage of RBCPS. Moreover, the active influx of Zn2+ into RBCs induced by their co-incubation with both ZnCl2 and A23187 did not increase the percent of RBCPS as compared to RBCs incubated with ZnCl2 alone. Taken together, these results demonstrate that the band-3 clustering agents (AO or ZnCl2) induce PS externalization in a Ca2+ independent manner, and we hereby suggest a possible scenario for this phenomenon.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Análise por Conglomerados , Eritrócitos/citologia , Fosfatidilserinas/metabolismo , Laranja de Acridina/farmacologia , Anexina A5/metabolismo , Cálcio/farmacologia , Senescência Celular , Cloretos/farmacologia , Humanos , Compostos de Zinco/farmacologia
17.
Arch Med Res ; 51(3): 187-193, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32111493

RESUMO

BACKGROUND: In this study, we aimed to determine synergistic apoptotic and cytotoxic effects of methylstat and bortezomib on U266 and ARH77 multiple myeloma (MM) cells. METHODS: Cytotoxic effects of the drugs were demonstrated by MTT cell proliferation assay while apoptotic effects were examined by loss of mitochondrial membrane potential (MMP) by JC-1 MMP detection kit, changes in caspase-3 enzyme activity and Annexin-V apoptosis assay by flow cytometry. Expression levels of apoptotic and antiapoptotic genes were examined by qRT-PCR. RESULTS: Our results showed that combination of methylstat and bortezomib have synergistic antiproliferative effect on MM cells as compared to either agent alone. These results were also confirmed by showing synergistic apoptotic effects determined by increased loss of mitochondrial membrane potential and increased caspase-3 enzyme activity and relocation of phosphotidyleserine on the cell membrane by Annexin-V/PI double staining. Combination of bortezomib with methylstat arrested cells at the S phase of the cell cycle. Methylstat treatment caused upregulation of FASLG, NGFR, TNF, TNFRS10B and TNFRS1B apoptotic genes and downregulation of AKT1, AVEN, BAG1 BCL2L2 and RELA antiapoptotic genes in a dose and time dependent manner. CONCLUSION: In conclusion, our data suggested that bortezomib in combination with methylstat decreased cell proliferation and induced apoptosis significantly in U266 and ARH77 cells. When supported with in vivo analyses, methylstat might be considered as a potential new agent for the treatment of MM.


Assuntos
Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Proliferação de Células/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Anexina A5/metabolismo , Antineoplásicos/farmacologia , Bortezomib/metabolismo , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo
18.
Nat Commun ; 11(1): 1137, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111835

RESUMO

The interaction between immune cells and phosphatidylserine (PS) molecules exposed on the surface of apoptotic-tumor bodies, such as those induced by chemotherapies, contributes to the formation of an immunosuppressive tumor microenvironment (TME). Annexin A5 (AnxA5) binds with high affinity to PS externalized by apoptotic cells, thereby hindering their interaction with immune cells. Here, we show that AnxA5 administration rescue the immunosuppressive state of the TME induced by chemotherapy. Due to the preferential homing of AnxA5 to the TME enriched with PS+ tumor cells, we demonstrate in vivo that fusing tumor-antigen peptide to AnxA5 significantly enhances its immunogenicity and antitumor efficacy when administered after chemotherapy. Also, the therapeutic antitumor effect of an AnxA5-peptide fusion can be further enhanced by administration of other immune checkpoint inhibitors. Our findings support the administration of AnxA5 following chemotherapy as a promising immune checkpoint inhibitor for cancer treatment.


Assuntos
Anexina A5/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Fatores Imunológicos/uso terapêutico , Neoplasias/terapia , Animais , Anexina A5/genética , Anexina A5/metabolismo , Anticorpos Bloqueadores/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Linhagem Celular Tumoral , Cisplatino/efeitos adversos , Cisplatino/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/uso terapêutico , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
19.
Biomed Res Int ; 2020: 9478789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32076621

RESUMO

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely.


Assuntos
Pressão Hidrostática , Neoplasias Cutâneas/patologia , Pele/patologia , Anexina A5 , Apoptose , Proliferação de Células , Epiderme/patologia , Matriz Extracelular/patologia , Fibroblastos/patologia , Humanos , Queratinócitos/patologia , Melanócitos , Nevo Pigmentado
20.
Nat Commun ; 11(1): 230, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932647

RESUMO

Annexins are abundant cytoplasmic proteins, which bind to membranes that expose negatively charged phospholipids in a Ca2+-dependent manner. During cell injuries, the entry of extracellular Ca2+ activates the annexin membrane-binding ability, subsequently initiating membrane repair processes. However, the mechanistic action of annexins in membrane repair remains largely unknown. Here, we use high-speed atomic force microscopy (HS-AFM), fluorescence recovery after photobleaching (FRAP), confocal laser scanning microscopy (CLSM) and molecular dynamics simulations (MDSs) to analyze how annexin-V (A5) binds to phosphatidylserine (PS)-rich membranes leading to high Ca2+-concentrations at membrane, and then to changes in the dynamics and organization of lipids, eventually to a membrane phase transition. A5 self-assembly into lattices further stabilizes and likely structures the membrane into a gel phase. Our findings are compatible with the patch resealing through vesicle fusion mechanism in membrane repair and indicate that A5 retains negatively charged lipids in the inner leaflet in an injured cell.


Assuntos
Anexina A5/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Anexina A5/química , Cálcio/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Fusão de Membrana , Microscopia de Força Atômica , Microscopia Confocal , Simulação de Dinâmica Molecular , Transição de Fase , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Agregados Proteicos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...