952 resultados para HUMAN RED-CELLS
Resumo:
RNA interference (RNAi) has been shown to be a valuable tool to specifically target gene expression in a number of organisms becoming an indispensable weapon in the arsenal in functional genomics. In this study, we demonstrate that streptolysin-O (SLO) reversible permeabilisation is an efficient method to deliver small interfering RNAs (siRNAs) to hard-to-transfect human myeloma cell lines. We used published, pre-validated siRNAs for ERK2 and non-silencing siRNA control. We transfected siRNAs into human myeloma cell lines using SLO reversible permeabilisation method. Flow cytometry and western blot analysis were performed to assess the effect of SLO on transfection efficiency and ERK2 knockdown. These experiments demonstrate that SLO reversible permeabilisation method is an efficient and easy-to-use method to deliver siRNAs into human myeloma cell lines. Optimised SLO permeabilisation method showed to transfect >80% of JIM-3, H929, RPM18226 and U266 cells, with minimal effect on cell viability (<10%) and cell cycle. Equally important, SLO permeabilisation induced a substantial knockdown of ERK2 at the protein level. These studies demonstrate that reversible SLO permeabilisation can successfully be applied to hard-to-transfect human myeloma cell lines to effectively silence genes. (C) 2008 Published by Elsevier B.V.
Resumo:
Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.
Resumo:
Acute myeloid leukemia (AML) involves the proliferation, abnormal survival and arrest of cells at a very early stage of myeloid cell differentiation. The biological and clinical heterogeneity of this disease complicates treatment and highlights the significance of understanding the underlying causes of AML, which may constitute potential therapeutic targets, as well as offer prognostic information. Tribbles homolog 2 (Trib2) is a potent murine oncogene capable of inducing transplantable AML with complete penetrance. The pathogenicity of Trib2 is attributed to its ability to induce proteasomal degradation of the full length isoform of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα p42). The role of TRIB2 in human AML cells, however, has not been systematically investigated or targeted. Across human cancers, TRIB2 oncogenic activity was found to be associated with its elevated expression. In the context of AML, TRIB2 overexpression was suggested to be associated with the large and heterogeneous subset of cytogenetically normal AML patients. Based upon the observation that overexpression of TRIB2 has a role in cellular transformation, the effect of modulating its expression in human AML was examined in a human AML cell line that expresses high levels of TRIB2, U937 cells. Specific suppression of TRIB2 led to impaired cell growth, as a consequence of both an increase in apoptosis and a decrease in cell proliferation. Consistent with these in vitro results, TRIB2 silencing strongly reduced progression of the U937 in vivo xenografts, accompanied by detection of a lower spleen weight when compared with mice transplanted with TRIB2- expressing control cells. Gene expression analysis suggested that TRIB2 modulates apoptosis and cell-cycle sensitivity by influencing the expression of a subset of genes known to have implications on these phenotypes. Furthermore, TRIB2 was found to be expressed in a significant subset of AML patient samples analysed. To investigate whether increased expression of this gene could be afforded prognostic significance, primary AML cells with dichotomized levels of TRIB2 transcripts were evaluated in terms of their xenoengraftment potential, an assay reported to correlate with disease aggressiveness observed in humans. A small cohort of analysed samples with higher TRIB2 expression did not associate with preferential leukaemic cell engraftment in highly immune-deficient mice, hence, not predicting for an adverse prognosis. However, further experiments including a larger cohort of well characterized AML patients would be needed to clarify TRIB2 significance in the diagnostic setting. Collectively, these data support a functional role for TRIB2 in the maintenance of the oncogenic properties of human AML cells and suggest TRIB2 can be considered a rational therapeutic target. Proteasome inhibition has emerged as an attractive target for the development of novel anti-cancer therapies and results from translational research and clinical trials support the idea that proteasome inhibitors should be considered in the treatment of AML. The present study argued that proteasome inhibition would effectively inhibit the function of TRIB2 by abrogating C/EBPα p42 protein degradation and that it would be an effective pharmacological targeting strategy in TRIB2-positive AMLs. Here, a number of cell models expressing high levels of TRIB2 were successfully targeted by treatment with proteasome inhibitors, as demonstrated by multiple measurements that included increased cytotoxicity, inhibition of clonogenic growth and anti-AML activity in vivo. Mechanistically, it was shown that block of the TRIB2 degradative function led to an increase of C/EBPα p42 and that response was specific to the TRIB2-C/EBPα axis. Specificity was addressed by a panel of experiments showing that U937 cells (express detectable levels of endogenous TRIB2 and C/EBPα) treated with the proteasome inhibitor bortezomib (Brtz) displayed a higher cytotoxic response upon TRIB2 overexpression and that ectopic expression of C/EBPα rescued cell death. Additionally, in C/EBPα-negative leukaemia cells, K562 and Kasumi 1, Brtz-induced toxicity was not increased following TRIB2 overexpression supporting the specificity of the compound on the TRIB2-C/EBPα axis. Together these findings provide pre-clinical evidence that TRIB2- expressing AML cells can be pharmacologically targeted with proteasome inhibition due, in part, to blockage of the TRIB2 proteolytic function on C/EBPα p42. A large body of evidence indicates that AML arises through the stepwise acquisition of genetic and epigenetic changes. Mass spectrometry data has identified an interaction between TRIB2 and the epigenetic regulator Protein Arginine Methyltransferase 5 (PRMT5). Following assessment of TRIB2‟s role in AML cell survival and effective targeting of the TRIB2-C/EBPα degradation pathway, a putative TRIB2/PRMT5 cooperation was investigated in order to gain a deeper understanding of the molecular network in which TRIB2 acts as a potent myeloid oncogene. First, a microarray data set was interrogated for PRMT5 expression levels and the primary enzyme responsible for symmetric dimethylation was found to be transcribed at significantly higher levels in AML patients when compared to healthy controls. Next, depletion of PRMT5 in the U937 cell line was shown to reduce the transformative phenotype in the high expressing TRIB2 AML cells, which suggests that PRMT5 and TRIB2 may cooperate to maintain the leukaemogenic potential. Importantly, PRMT5 was identified as a TRIB2-interacting protein by means of a protein tagging approach to purify TRIB2 complexes from 293T cells. These findings trigger further research aimed at understanding the underlying mechanism and the functional significance of this interplay. In summary, the present study provides experimental evidence that TRIB2 has an important oncogenic role in human AML maintenance and, importantly in such a molecularly heterogeneous disease, provides the rational basis to consider proteasome inhibition as an effective targeting strategy for AML patients with high TRIB2 expression. Finally, the identification of PRMT5 as a TRIB2-interacting protein opens a new level of regulation to consider in AML. This work may contribute to our further understanding and therapeutic strategies in acute leukaemias.
Resumo:
Background: The emergence of multiple-drug resistance bacteria has become a major threat and thus calls for an urgent need to search for new effective and safe anti-bacterial agents. Objectives: This study aims to evaluate the anticancer and antibacterial activities of secondary metabolites from Penicillium sp. , an endophytic fungus associated with leaves of Garcinia nobilis . Methods: The culture filtrate from the fermentation of Penicillium sp. was extracted and analyzed by liquid chromatography– mass spectrometry, and the major metabolites were isolated and identified by spectroscopic analyses and by comparison with published data. The antibacterial activity of the compounds was assessed by broth microdilution method while the anticancer activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: The fractionation of the crude extract afforded penialidin A-C (1-3), citromycetin (4), p-hydroxyphenylglyoxalaldoxime (5) and brefelfin A (6). All of the compounds tested here showed antibacterial activity (MIC = 0.50 – 128 μg/mL) against Gramnegative multi-drug resistance bacteria, Vibrio cholerae (causative agent of dreadful disease cholera) and Shigella flexneri (causative agent of shigellosis), as well as the significant anticancer activity (LC50 = 0.88 – 9.21 μg/mL) against HeLa cells. Conclusion: The results obtained indicate that compounds 1-6 showed good antibacterial and anticancer activities with no toxicity to human red blood cells and normal Vero cells.
Resumo:
Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016
Resumo:
Purpose: To investigate the effect of Allium sativum (garlic) methanol extract on viability and apoptosis of human leukemic cells. Methods: Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ug/mL of Allium sativum extract following 48-h treatment on U-937, Jurkat Clone E6-1 and K-562 cell lines. The mode of cell death was determined by Annexin V-FITC staining and analyzed by flow cytometry. Results: The results show that the half-maximal inhibitory concentration (IC50) of A. sativum on U-937, Jurkat Clone E6-1, K-562 cell lines was 105 ± 2.21, 489 ± 4.51 and 455 ± 3.13 μg/mL, respectively, compared with negative control, while apoptosis was 17.93 ± 0.95 % for U-937 cells (p ≤ 0.05), 38.37 ± 1.88 % for Jurkat Clone E6-1 cells (p ≤ 0.001) and 16.37 ± 1.10 % for K-562 cells. A majority of the cells were inhibited by the extract via apoptosis. Only U-937 cells (6.87 ± 0.65 %) showed significant necrosis compared to negative control (p ≤ 0.05). Conclusion: K-562 cells are the most resistant against garlic extract, in contrast to Jurkat Clone E6-1 cells. Garlic extract does not induce necrosis in Jurkat Clone E6-1 and K-562 cells.
Resumo:
Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.
Resumo:
A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.
Resumo:
Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.
Resumo:
The aim of the present study was to perform an in vitro analysis of the antimicrobial and antiproliferative potential of an extract from Anadenanthera colubrina (Vell.) Brenan (angico) and chemically characterize the crude extract. Antimicrobial action was evaluated based on the minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration, and the inhibition of formation to oral biofilm. Cell morphology was determined through scanning electron microscopy (SEM). Six strains of tumor cells were used for the determination of antiproliferative potential. The extract demonstrated strong antifungal activity against Candida albicans ATCC 18804 (MIC = 0.031 mg/mL), with similar activity found regarding the ethyl acetate fraction. The extract and active fraction also demonstrated the capacity to inhibit the formation of Candida albicans to oral biofilm after 48 hours, with median values equal to or greater than the control group, but the difference did not achieve statistical significance (P > 0.05). SEM revealed alterations in the cell morphology of the yeast. Regarding antiproliferative activity, the extract demonstrated cytostatic potential in all strains tested. The present findings suggest strong antifungal potential for Anadenanthera colubrina (Vell.) Brenan as well as a tendency toward diminishing the growth of human tumor cells.
Resumo:
The aim of this study was to evaluate the degree of conversion (DC) and the cytotoxicity of photo-cured experimental resin composites containing 4-(N,N-dimethylamino)phenethyl alcohol (DMPOH) combined to the camphorquinone (CQ) compared with ethylamine benzoate (EDAB). The resin composites were mechanically blended using 35 wt% of an organic matrix and 65 wt% of filler loading. To this matrix was added 0.2 wt% of CQ and 0.2 wt% of one of the reducing agents tested. 5x1 mm samples (n=5) were previously submitted to DC measurement and then pre-immersed in complete culture medium without 10% (v/v) bovine serum for 1 h or 24 h at 37 °C in a humidifier incubator with 5% CO2 and 95% humidity to evaluate the cytotoxic effects of experimental resin composites using the MTT assay on immortalized human keratinocytes cells. As a result of absence of normal distribution, the statistical analysis was performed using the nonparametric Kruskal-Wallis to evaluate the cytotoxicity and one-way analysis of variance to evaluate the DC. For multiple comparisons, cytotoxicity statistical analyses were submitted to Student-Newman-Keuls and DC analysis to Tukey's HSD post-hoc test (=0.05). No significant differences were found between the DC of DMPOH (49.9%) and EDAB (50.7%). 1 h outcomes showed no significant difference of the cell viability between EDAB (99.26%), DMPOH (94.85%) and the control group (100%). After 24 h no significant difference were found between EDAB (48.44%) and DMPOH (38.06%), but significant difference was found compared with the control group (p>0.05). DMPOH presented similar DC and cytotoxicity compared with EDAB when associated with CQ.
Resumo:
In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investigated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a high selectivity with potent growth inhibition and cell death for the glioma cell line-the most aggressive CNS cancer.
Resumo:
Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-β1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.
Resumo:
Arrabidaea chica (H&B) Verlot is a plant popularly known as Pariri and this species is a known source of anthocyanins, flavonoids and tannins. This report describes an approach involving enzymatic treatment prior to extraction procedures to enhance A chica crude extract anticancer activity. Anticancer activity in human cancer cell lines in vitro using a 48 h SRB cell viability assay was performed to determine growth inhibition and cytotoxic properties. The final extraction yield without enzyme treatment was higher (24.28%) compared to the enzyme-treated material (19.03%), with an enhanced aglycones anthocyanin ratio as determined by HPLC- DAD and LC-MS with direct infusion.
Resumo:
Em 1999, as células-tronco foram eleitas "Scientific Breakthrough of the Year" (avanço científico do ano) pela revista Science¹. Naquele ano, foi demonstrado que células-tronco de tecidos adultos mantinham a capacidade de se diferenciar em outros tipos de tecidos. No ano anterior, as primeiras linhagens de células-tronco embrionárias humanas foram estabelecidas. Desde então, o número de artigos científicos sobre células-tronco vem crescendo exponencialmente, onde novos paradigmas são estabelecidos. Neste artigo, farei uma revisão da área de células-tronco com um foco especial em seu uso como agente terapêutico em doenças comuns como diabetes e cardiopatias. As células-tronco serão tratadas em dois grupos distintos: as embrionárias e as adultas. Enquanto o potencial de diferenciação das primeiras está bem caracterizado em camundongos e em humanos, seu uso em terapia celular e em pesquisa tem sido dificultado por questões de histocompatibilidade, segurança e ética. Em contraste, células-tronco adultas não apresentam estes empecilhos, apesar da extensão de sua plasticidade ainda estar sob investigação. Mesmo assim, diversos testes clínicos em humanos estão em andamento utilizando células-tronco adultas, principalmente derivadas da medula óssea. Discutirei ainda a importância de se trabalhar com as duas classes de células-tronco humanas de forma a se cumprir suas promessas terapêuticas.