Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent ...and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.
Propolis extracts are widely used in traditional folk medicine and exhibit several properties such as antitumor, anti-inflammatory, and antimicrobial. However, these products have not been ...investigated in combination with medicines used in clinical practice.
This study aimed to evaluate the chemical composition of propolis extracts from Apis mellifera scutellata and different Meliponini species and characterize their cytotoxicity against tumor cells, antibacterial effects, and interference with the actions of doxorubicin and gentamicin.
Chromatographic and spectrometric analyses were performed using ultra-high-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS). Propolis extracts were evaluated for cytotoxicity and synergism using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the antimicrobial activity was examined using the broth microdilution technique and synergism was investigated using checkerboard and time-kill assays.
The chemical characterization revealed the presence of 63 compounds, and the extracts showed selective cytotoxicity against tumor cell lines. Propolis extracts of mandaçaia and mirim exerted selective synergistic cytotoxicity in combination with doxorubicin. Except for the tubuna extract, all evaluated extracts exhibited antibacterial effects on gram-positive strains. Mandaçaia and mirim extracts exerted a synergistic effect with gentamicin; however, only mandaçaia extract exerted a selective effect.
Propolis could be a source of antineoplastics and antibiotics. These natural products may reduce the occurrence of doxorubicin and gentamicin related adverse effects, resistance, or both.
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•63 compounds were identified in propolis extracts.•Propolis extracts exert selective synergistic cytotoxicity with doxorubicin.•Propolis extracts have antibacterial action.•Propolis extract exerts selective synergistic effect with gentamicin.
Cancer is an inflammatory disease because carcinogenesis and tumor progression depend on intrinsic and extrinsic inflammatory pathways. Although species of the genus Aspidosperma are widely used to ...treat tumors, and there is ethnopharmacological evidence for traditional use of the species A. subincanum as an anti-inflammatory agent, its antineoplastic potential is unknown.
To evaluate toxic effects of the indole alkaloid–rich fraction (IAF) of A. subincanum on the MCF7 cell line and identify some of the anti-inflammatory mechanisms involved.
Chromatographic analyses were performed by ultra-high-performance liquid chromatography with electrospray ionization mass spectrometry, and cytotoxic and antiproliferative effects of IAF were verified by MTT and clonogenic assays. Cell cycle alterations were analyzed by measuring DNA content, while propidium iodide and acridine orange staining was performed to determine the type of induced cell death. The expression of apoptosis markers and proteins involved in cell proliferation and survival pathways was analyzed by immunoblotting, RT-qPCR, and ELISAs. Interference with redox status was investigated using a DCFH-DA probe and by measuring catalase activity.
Chromatographic analyses showed that IAF is a complex mixture containing indole alkaloids. IAF selectively exerted toxic and antiproliferative effects, elevating the Bax/Bcl-xL ratio and inducing apoptosis in MCF7 cells. IAF decreased intracellular reactive oxygen species levels and increased catalase activity, while reducing the IL-8 level and suppressing COX-2 expression.
IAF induces apoptosis in MCF7 cells by suppressing COX-2 expression while reducing IL-8 levels and intracellular content of reactive oxygen species.
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•Four indolic alkaloids were proposed in IAF.•IAF exerts cytotoxic and antiproliferative effects in MCF7 cells.•IAF induces apoptosis in MCF7 cells.•IAF's cytotoxicity and antiproliferative action involves modulation of redox status.•The effects of IAF in MCF7 cells are related to modulation of inflammatory pathways.
Studies have demonstrated that diet rich in cruciferous vegetables of the Brassicaceae family can reduce the risk of cardiovascular diseases and oxidative stress levels. Nasturtium officinale ...(Brassicaceae), commonly known as watercress is a perennial dicotyledonous plant usually found close to water. Although previous investigations have demonstrated the beneficial effects of watercress on hypercholesterolemia in animal studies, until now no such studies have been conducted with humans, up to this time. This study aimed to investigate whether overweight individuals were able to improve or maintain their serum lipid and oxidative stress markers when given standardized extract of Nasturtium officinale (SENO) as a supplement. This was a randomized, double‐blind, and placebo‐controlled trial conducted over 5 weeks. Thirty‐four overweight people with physical disabilities were selected randomly to participate in this study and then they were assigned randomly to two groups, one treated with 750 mg//kg/d of SENO and the other treated with 750 mg/kg/d of placebo. The results indicated that SENO caused a significant improvement in the levels of low‐density lipoprotein cholesterol, creatinine, and lipid peroxidation. However, SENO did not cause a significant statistical change in total serum cholesterol, triacylglycerol, and high‐density lipoprotein levels; catalase, superoxide dismutase, creatinine, alanine aminotransferase, aspartate aminotransferase, and urea parameters. The present data might provide supportive evidence that SENO did not cause any harm and positively affected low‐density lipoprotein cholesterol profile and creatinine as well as lipid peroxidation levels in the participants. Nevertheless, further studies are suggested to clarify the results presented in this clinical trial.
It is well established that plants from the Brassicaceae family, particularly watercress, have been associated to reduce oxidative DNA damage. Nasturtium officinale R. Br (watercress) contains ...glucosinolates, with anti‐inflammatory action and protective effect on human health against oxidative stress. We aimed to evaluate whether the standardized extract of Nasturtium officinale (SENO) is capable of changing biomarkers of oxidative stress and inflammation in people with physical disabilities. 65 people enrolled this study: as a control group composed by; 15 people with no physical disability assessed once, 25 people with physical disabilities using 750 mg/kg/day of SENO, and 25 people with physical disabilities using 750 mg/kg/day of placebo‐control for 5 weeks. Biomarkers of oxidative stress and inflammation were analyzed on day 0 and 36. The results indicated that SENO was associated with decreasing levels of lipid peroxidation, protein carbonyl, catalase, superoxide dismutase, and C‐reactive protein. Furthermore, the cytokine kit demonstrated below and out of invertible range, which was impossible to detect the inflammatory process. Despite the cytokine kit was not able to detect the inflammation; these data might provide supportive evidence that SENO, have affected positively people with physical disabilities decreasing their biomarkers of oxidative stress and C‐reactive protein. Further studies are required.
Supercritical fluid technologies offer an innovative method for food industry and drug discovery from natural sources. The aim of the study is to investigate the anti-tumor activity of piperine rich ...extract by supercritical fluid (SFE) from black pepper (Piper nigrum). In silico docking simulations predicted anti-tumor molecular mechanism and protein-piperine hydrophobic interactions, showing hydrogen bonds between piperine and residue Ser5 inside the ATP binding site in CDK2. Moreover, piperine interacts with peptide substrate residue Lys8 inside its binding site in Cyclin A molecule. Other predicted interaction showed piperine inside the hydrophobic groove of Bcl-xL. Confirming the docking simulation, in vitro assays with SFE (40 °C/30 MPa) showed cytotoxicity to MCF-7 cells (IC50 = 27.8 ± 6.8 μg/ml) correlated to increased apoptosis. Balb/c mice-bearing Ehrlich Ascites Carcinoma (EAC) group that received the SFE (100 mg/kg/day) showed tumor growth inhibition (60%) and increased mice survival (50%), probably related to cell cycle arrest (G2/M) and increased apoptosis. In vivo treatments with SFE increased the expression of pro-apoptotic proteins (p53 and Bax), inhibited cell cycle proteins (CDK2, Cyclin A) and anti-apoptotic protein (Bcl-xL). Thus, confirming in silico predicted inhibitory interactions. These results clearly showed promising performance of the piperine-rich fraction recovered from black pepper, drawing attention to its use as complementary therapy for cancer.
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•Millenary nourish traditions used black pepper (Piper nigrum) as flavoring and antimicrobial has an anti-tumor effect.•Docking predicted interactions between piperine - protein (CDK2-cyclin A or Bcl-xL).•Predicted in silico interactions between piperine-protein key were H-bonds and hydrophobic interactions.•Piper nigrum supercritical dioxide carbon extract was cytotoxic and antiproliferative against breast tumor cell MCF-7.•Piper nigrum Supercritical extract inhibited in vivo Ehrlich Carcinoma increasing apoptosis and mice survival.
Although cancer is a chronic disease, most of the in vitro experiments to assess the effectiveness of intervention are performed in hours or a few days. Moreover, none of the available methodologies ...to measure cell proliferation are adapted to provide information about the growth kinetic during and after treatment. Thus, the objective of this work is to provide a guide to assess long-term changes in cell population size to be used mainly in cancer research. Cumulative population doubling (CPD) graphs based on cell counting for in vitro or tumor volume for in vivo assays were used to calculate four parameters: relative end CPD (RendCPD), to quantify the end point analysis of proliferation; relative area under curve (rAUC), to describe the global chronic effect of a treatment; relative time to cross a threshold (RTCT), to indicate the delay in cell population recovery produced by a treatment; and relative proliferation rate (RPR), to describe the relative regrowth velocity of the cells that survived after treatment. These parameters describe not only the acute and chronic effects of a treatment but also the behavior of cells that are not eliminated by the treatment, providing crucial information about the growth kinetic of the surviving population. Moreover, the proposed analysis allowed the grouping of independent CPD experiments quantified at different time points and even the direct comparison of in vitro and in vivo experiments. Therefore, this new way to analyze long-term outcomes provides a global view of the effectiveness of an intervention, as an important tool for long-term studies.
The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects ...on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.
Biodiesel is a biofuel produced by the esterification reaction. It is an option to replace fossil fuels, mainly diesel, due to its similar physical and chemical properties. In this research, a ...mathematical modeling was performed to predict the antioxidant mass to be added in a commercial biodiesel to achieve the minimum oxidative stability provided by the legislation. An equation for each additive was proposed, with the mass of these antioxidants considered as dependent variables and the conformity parameters as independent ones, obtaining multiple regression equations without intercept. The BHA and BHT antioxidants were used. The equations provided were lower at the 5% level and presented determination coefficients (R²) equal to 0.9790 and 0.9770 for the BHA and BHT, respectively, proving their predictability ability and also showing that they can be used to estimate the antioxidant mass to be added in a biodiesel to reach the current regulations.
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