RESUMEN
INTRODUCTION: Research in tumor treatment has shown promising results using extracellular vesicles (EVs) derived from immune cells. EVs derived from M1 macrophages (proinflammatory), known as M1-EVs, have properties that suppress tumor growth, making them a promising treatment tool for immune susceptible tumors such as melanoma. Here, small unaltered M1-EVs (M1-sEVs) were employed in a 3D mouse melanoma model (melanospheres) to evaluate such activity. METHODS: Macrophages were polarized and EVs were isolated by ultracentrifugation. The EVs obtained were characterized based on size, with measurements performed by dynamic light scattering and electron microscopy, and the expression profiles of microRNAs were analyzed by microarray and PCR. Melanospheres were used to evaluate the cytotoxicity of M1-sEVs. Pondering a possible future transposition from the animal model to the human, human melanoma cells were transfected with a specific miRNA, and the impact on cell proliferation was evaluated. RESULTS: The isolated EVs showed a size distribution between 50-400 nm in diameter, but preeminently in a range of 70-90 nm. M1-sEVs demonstrated a remarkable ability to reduce cell proliferation and viability in the melanospheres, leading to a decrease in their volume. M1-sEVs contained unique miRNAs, including miR-29a-3p, which exhibited significant antitumor activities according to bioinformatics analysis. Validation of the antitumor effects of miR-29a-3p was obtained by a functional evaluation, i.e., by inducing miRNA overexpression in human melanoma cells (SK-MEL-28). CONCLUSION: Although further research would be advisable, the study provides evidence supporting the potential of M1-sEVs and their miRNA load as a possible targeted immune therapy for melanoma.
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Vesículas Extracelulares , Melanoma , MicroARNs , Animales , Humanos , Ratones , Melanoma/terapia , Modelos Animales de Enfermedad , Macrófagos , MicroARNs/genéticaRESUMEN
Senecio spp. is one of the most frequent plant-related poisonings in cattle. Its ingestion generates the disease seneciosis, characterized by hepatic damages. Liver biopsies and serum markers dosage are tools used in diagnosis; however, many breeding cattle are undiagnosed. MicroRNAs are non-coding RNA, stable in biological fluids. Their difference in expression levels may indicate the presence of the poisoning. We analyzed the miRNA profiling to identify potential diagnostic biomarkers for Senecio brasiliensis poisoning. The expression of miR-21, miR-885, miR-122, miR-181b, miR-30a, miR-378, and let-7 f were evaluated in the serum of exposed cattle. At least one histological change was found in liver and lower quantity of albumin and high AST and ALP were also detected. MiRNAs miR-30a, miR-378, miR-21, miR-885, and miR-122 presented significantly higher expression in intoxicated animals than in healthy animals. Furthermore, miR-122, miR-885, and, especially, miR-21 signatures demonstrated high sensitivity and specificity, with potential application for detecting poisoning.
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MicroARNs , Senecio , Animales , Biomarcadores , Bovinos , Hígado , MicroARNs/genética , Senecio/genéticaRESUMEN
The progression to a castration-resistant prostate cancer can occur after treatment with androgen deprivation therapy, resulting in poor prognosis and ineffective therapy response. Hormone dependence transition has been associated with increased tumor vascularization. Considering that exosomes are important components in communication between tumor cells and the microenvironment, we examined the angiogenic potential of exosomes released from Pca cell lines with distinctive profiles of androgen response through exosomes isolation, microscopy and uptake, functional assays follow up by microarray, RT-qPCR and bioinformatics analysis. HUVEC cells treated with PC-3 exosomes (androgen independent) showed increased invasion and tube formation ability. In order to identify microRNAs (miRNAs) related to the angiogenic response, the characterization of exosomal miRNA profile was performed. As result we suggest that the miR-27a-3p could be involved in the pro-angiogenic effect of PC-3 exosomes.
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Exosomas , MicroARNs , Neoplasias de la Próstata , Antagonistas de Andrógenos/metabolismo , Línea Celular Tumoral , Exosomas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias de la Próstata/patología , Microambiente TumoralRESUMEN
Prostate cancer-related deaths are mostly caused by metastasis, which indicates the importance of identifying clinical prognostic biomarkers. In this study, we evaluated the expression profile of exosomal microRNAs (miRNAs) derived from metastatic prostate cancer (mPCa) cell lines (LNCaP and PC-3). miRNA signatures in exosomes and cells were evaluated by miRNA microarray analysis. Fourteen miRNAs were identified as candidates for specific noninvasive biomarkers. The expression of five miRNAs was validated using RT-qPCR, which confirmed that miR-205-5p, miR-148a-3p, miR-125b-5p, miR-183-5p, and miR-425-5p were differentially expressed in mPCa exosomes. Bioinformatic analyses showed that miR-425-5p was associated with residual tumor, pathologic T and N stages, and TP53 status in PCa samples. Gene ontology analysis of negatively correlated and predicted targeted genes showed enrichment of genes related to bone development pathways. The LinkedOmics database indicated that the potential target HSPB8 has a significant negative correlation with miR-425-5p. In conclusion, this study identified a panel of exosomal miRNAs with potential value as prognostic biomarkers for prostate cancer.
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Exosomas , MicroARNs , Neoplasias de la Próstata , Biomarcadores/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biología Computacional , Exosomas/genética , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
Acute-on-chronic liver failure (ACLF) is a condition characterized by acute decompensation of cirrhosis, associated with organ failure(s), and high short-term mortality. The microRNAs or miRNAs are small non-coding RNA molecules, stable in circulating samples such as biological fluids, and the difference in expression levels may indicate the presence, absence and/or stage of the disease. We analyzed here the miRNA profiling to identify potential diagnostic or prognostic biomarkers for ACLF. The major miRNAs discovered were validated in a cohort of patients with acute decompensation of cirrhosis grouped in no ACLF or ACLF according to EASL-CLIF definition. Relationship between serum miRNAs and variables associated with liver-damage and survival outcomes were verified to identify possible prognostic markers. Our results showed twenty altered miRNAs between no ACLF and ACLF patients, and twenty-seven in patients who died in 30 days compared with who survived. In validation phase, miR-223-3p and miR-25-3p were significantly altered in ACLF patients and in those who died in 30 days. miR-223-3p and miR-25-3p expression were associated with the lowest survival in 30 days. The decrease in miR-223-3p and miR-25-3p expression was associated with the presence of ACLF and poor prognosis. Of these, miR-25-3p was independently related to ACLF and 30-day mortality.
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Insuficiencia Hepática Crónica Agudizada/mortalidad , Biomarcadores/sangre , Cirrosis Hepática/mortalidad , MicroARNs/genética , Insuficiencia Hepática Crónica Agudizada/sangre , Insuficiencia Hepática Crónica Agudizada/genética , Insuficiencia Hepática Crónica Agudizada/patología , Estudios de Casos y Controles , Femenino , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Puntuaciones en la Disfunción de Órganos , Pronóstico , Curva ROC , Índice de Severidad de la Enfermedad , Tasa de SupervivenciaRESUMEN
The present study characterized propolis extracts produced by Scaptotrigona bipunctata (Tubuna) and Melipona quadrifasciata (Mandaçaia) by LC-MS/MS; their cytotoxicity as well as the mechanism of action in a melanoma cellular model were also assessed. The chemical characterization performed by UPLC-ESI-QTOF/MS2 analysis revealed uncommon presence of piperidinic alkaloids in Tubuna's propolis extract together with C-glycopyranoside flavonoids. Mandaçaia's propolis collected in the same area rather presented terpenoids and flavonoids. Regarding the mechanism of cytotoxicity, propolis extracts increased the accumulation of reactive oxygen species (ROS), reduced the potential of mitochondrial membrane, induced a decrease in the proteins Bcl-2 and AKT-3 levels, and decreased melanoma cells' migration and invasion. Both propolis extracts induced apoptosis while only Mandaçaia's propolis extract induced cell cycle arrest in G2/M.
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Apoptosis/efectos de los fármacos , Melanoma/tratamiento farmacológico , Própolis/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Alcaloides/química , Alcaloides/farmacología , Animales , Abejas/fisiología , Brasil , Línea Celular Tumoral , Movimiento Celular , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Piperidinas/química , Piperidinas/farmacología , Própolis/química , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodosRESUMEN
Drugs containing the1,4-dihydropyridine (DHP) core have recently attracted attention concerning their antiparasitic effect against various species of Leishmania and Trypanosoma. This approach named drugs repositioning led to interesting results, which have prompted us to prepare 21 DHP's analogues. The 1,4-DHP scaffold was decorated with different function groups at tree points including the nitrogen atom (NH and N-phenyl), the aryl group attached to C-4 (various substituted aryl residues) and the carbon atoms 2 and 6 (bearing Ph or Me groups). Moreover, the products were evaluated for their cytotoxicity on three cancer and a non-tumoral cell lines. Only 6 of them were antiproliferative and their weak effect (CC50 comprised between 27 and 98µM) suggested these DHPs as good candidates against the intracellular amastigote forms of L. amazonensis and T. cruzi. L. amazonensis was sensitive to DHPs 5, 11 and 15 (IC50 values at 15.11, 45.70 and 53.13µM, respectively) while 12 of them displayed significant to moderate trypanocidal activities against T. cruzi. The best trypanocidal activities were obtained with compounds 2, 18 and 21 showing IC50 values at 4.95, 5.44, and 6.64µM, respectively. A part of the N-phenylated DHPs showed a better selectivity than their NH analogues towards THP-1 cells. 4-Chlorophenyl, 4-nitrophenyl and 3-nitrophenyl residues attached to the carbon atom 4 turned to be important sub-structures for the antitrypanosomal activity.
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Bloqueadores de los Canales de Calcio/farmacología , Leishmania mexicana/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/síntesis química , Línea Celular , Línea Celular Tumoral , Dihidropiridinas , Humanos , Células MCF-7 , Ratones , Células 3T3 NIH , Relación Estructura-Actividad , Tripanocidas/síntesis químicaRESUMEN
We investigated the effect of dehydroepiandrosterone sulfate (DHEAS) and 17beta-estradiol on NTPDase activity in fresh clinical (VP60) and long-term-grown (30236 ATCC) isolates of Trichomonas vaginalis followed by NTPDase gene transcriptional analysis. ATP hydrolysis was activated in vitro by 17beta-estradiol (0.01-1.0microM) in the VP60 isolate. Treatment for 2h with 17beta-estradiol (0.01-1microM) promoted an inhibition in nucleotide hydrolysis in the 30236 isolate whereas the 12h-treatment promoted an activation of nucleotide hydrolysis in both isolates. ADP hydrolysis was inhibited in vitro by 1.0-5.0microM DHEAS in the ATCC isolate. The treatment with DHEAS (0.01-1.0microM) for 2h inhibited ATP and ADP hydrolysis in VP60; however, during a 12h-treatment with DHEAS, nucleotide hydrolysis was inhibited in both isolates. Two NTPDase orthologous (NTPDaseA and NTPDaseB) were identified and the treatment with DHEAS for 12h was able to inhibit mRNA NTPDaseA transcript levels from the VP60. These findings demonstrate that NTPDase activity and gene expression pattern are modulated by exposure to steroids in T. vaginalis.
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Sulfato de Deshidroepiandrosterona/farmacología , Estradiol/farmacología , Estrógenos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Nucleósido-Trifosfatasa/metabolismo , Trichomonas vaginalis/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Humanos , Hidrólisis/efectos de los fármacos , Cinética , Ratones , Datos de Secuencia Molecular , Nucleósido-Trifosfatasa/química , Nucleósido-Trifosfatasa/efectos de los fármacos , Nucleósido-Trifosfatasa/genética , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Trichomonas vaginalis/enzimología , Trichomonas vaginalis/genética , Trichomonas vaginalis/crecimiento & desarrolloRESUMEN
Trichomonas vaginalis infection may be influenced by the vaginal concentrations of estrogens. We have investigated the effects of 17beta-estradiol and dehydroepiandrosterone sulfate (DHEAS) on the ecto-5'-nucleotidase activity in fresh clinical (VP60) and in long-term-grown (30236 ATCC) isolates of T. vaginalis. In vitro exposure to DHEAS and 17beta-estradiol did not induce any changes in adenosine monophosphate (AMP) hydrolysis in these isolates. The treatment of parasites in the presence of DHEAS (0.01-1.0 microM) for 2 h inhibited AMP hydrolysis in VP60 isolate, whereas there were no significant changes in nucleotide hydrolysis in the presence of 17beta-estradiol. DHEAS and 17beta-estradiol (0.01-1.0 microM) for 2 h inhibited AMP hydrolysis in 30236 isolate. The 12 treatment with 0.1 microM DHEAS inhibited AMP hydrolysis, whereas 17beta-estradiol did not alter the nucleotide hydrolysis in VP60 isolate. Our findings have shown that the complex effect of steroid hormones and their receptors on T. vaginalis may promote changes in ecto-5'-nucleotidase activity during exposure to these hormones.