Anticancer activities of Brachydin C in human prostate tumor cells (DU145) grown in 2D and 3D models: Stimulation of cell death and downregulation of metalloproteinases in spheroids.

Brachydin C (BrC) has demonstrated in vitro cytotoxic and antiproliferative effects in prostate cancer cells. In the present study, we compare the anticancer effects of BrC in DU145 cells grown in common bidimensional cultures (2D) and multicellular tumor spheroids (MCTS), often denominated 3D in vitro models, that can better mimic the microenvironment of tissues. BrC IC50 values obtained in the resazurin assay after 24 h of treatment were 47.31 µM (2D) and 229.8 µM (3D) and these cytotoxic effects were time-dependent only in 3D. BrC (5.0-60 µM) interfered with the growth of MCTS and reduced cell viability after 11 days of treatment, a result that is not attributable to oxidative stress evaluated using the CM-H2 DCFDA probe. BrC (6.0 µM) impaired horizontal (wound-healing) and vertical cell migration and invasion (transwell assay) in 2D and BrC (5.0-60 µM) in 3D (ECM Gel®). BrC modulated the expression of genes BIRC5, TNF-α, CASP3, NKX3.1, MMP9, MMP11, CDH1, and ITGAM and downregulated proteins CASP7, BAX, and TNF-α in Western blotting analysis. In conclusion, BrC stimulated cell death and decreased epithelial-mesenchymal transition. Furthermore, DU145 MCTS displayed higher resistance to BrC-induced cell death than 2D cultures, a difference that should be considered in future approaches in prostatic cancer studies.

Flavone cirsimarin impairs cell proliferation, migration, and invasion in MCF-7 cells grown in 2D and 3D models.

The present study investigates the mechanisms underlying the in vitro antitumoral activity of cirsimarin (CIR 10 to 320 µM), a flavone extracted from the aerial parts of Scoparia dulcis L., on MCF-7 cells cultured in 2D and multicellular tumor spheroids (3D). CIR (from 40 µM) decreased cell viability in the resazurin assay and colony formation in the 2D model. In the same way, in the 3D model, CIR (from 40 µM) induced cell death (triple staining assay) and decreased spheroid integrity after 16 days with no induction of intracellular reactive species (CM-H2DCFDA). In 2D, CIR decreased the invasion (transwell) and horizontal migration (wound healing), while in 3D, CIR diminished cell migration (ECM® gel) and induced DNA damage (comet assay) possibly related to cell death. CIR mediated antitumoral effects in 3D spheroids by negative modulation of genes associated with cell proliferation (CCND1, CCNA2, CDK2, CDK4, and TNF) and death (BCL-XL, BAX, CASP9, and BIRC5). BIRC5 and CDKs inhibitors have been proposed as versatile anticancer drugs, which makes our results quite interesting. TNF negative modulation may also be related to the downregulation of MMP9 and MMP11 and anti-migration/invasion of MCF-7 cells cultured in 2D and 3D models. These are relevant properties for long-term strategies to avoid metastasis and improve the prognosis of breast cancer.

The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death.

Metastatic prostate cancer (mPCa) is resistant to several chemotherapeutic agents. Brachydin A (BrA), a glycosylated flavonoid extracted from Fridericia platyphylla, displays a remarkable antitumoral effect against in vitro mPCa cells cultured as bidimensional (2D) monolayers. Considering that three-dimensional (3D) cell cultures provide a more accurate response to chemotherapeutic agents, this study investigated the antiproliferative/antimetastatic effects of BrA and the molecular mechanisms underlying its action in mPCa spheroids (DU145) in vitro. BrA at 60-100 µM was cytotoxic, altered spheroid morphology/volume, and suppressed cell migration and tumor invasiveness. High-content analysis revealed that BrA (60-100 µM) reduced mitochondrial membrane potential and increased apoptosis and necrosis markers, indicating that it triggered cell death mechanisms. Molecular analysis showed that (i) 24-h treatment with BrA (80-100 µM) increased the protein levels of DNA disruption markers (cleaved-PARP and p-γ-H2AX) as well as decreased the protein levels of anti/pro-apoptotic (BCL-2, BAD, and RIP3K) and cell survival markers (p-AKT1 and p-44/42 MAPK); (ii) 72-h treatment with BrA increased the protein levels of effector caspases (CASP3, CASP7, and CASP8) and inflammation markers (NF-kB and TNF-α). Altogether, our results suggest that PARP-mediated cell death (parthanatos) is a potential mechanism of action. In conclusion, BrA confirms its potential as a candidate drug for preclinical studies against mPCa.

Anti-Ehrlichia properties of the dichloromethane fraction of Ageratum conyzoides associated with doxycycline: In vitro study / Propriedades anti-Ehrlichia da fração diclorometânica de Ageratum conyzoides associada com doxiciclina: estudo in vitro

ABSTRACT: The increasing number of cases of canine ehrlichiosis caused by Ehrlichia canis in hospitals and veterinary clinics has demonstrated the need for a new drug protocol for this disease. Doxycycline is used to treat ehrlichiosis, but the resistance of the microorganism to this treatment protocol, as well as the various side effects to the animals, has become a concern. Several studies have shown a positive interaction with extracts of plants and drugs, which allow for the reduction of the concentration necessary to produce the desired effect, minimizing adverse effects. This study determined the efficiency of the combination of the dichloromethane (DCM) fraction of Ageratum conyzoides L. with anti-Ehrlichia activity and doxycycline by using the checkerboard assay. Plant material was collected in São Luís, northeastern Brazil, followed by extraction in MeOH: H2O (8:2) and partitioning of the DCM fraction. After determining the Minimum Inhibitory Concentration (MIC) of the fraction under study against DH82 cells infected with Ehrlichia canis, it was combined with doxycycline to derive the Fractional Inhibitory Concentration Index (CIF Index). A reduction of 5.83 times the doxycycline minimum inhibitory concentration was observed, showing that this fraction of A. conyzoides composed predominantly by the class of lignans, identified by mass spectrometry notably intensified the activity of doxycycline against E. canis, resulting in a synergistic effect.

RESUMO: O crescente número de casos de erliquiose canina por Ehrlichia canis em hospitais e clínicas veterinárias tem demonstrado a necessidade de um novo protocolo de medicamentos para essa doença. A doxiciclina é usada para tratar a erliquiose, mas a resistência do microrganismo a esse protocolo de tratamento, bem como os diversos efeitos colaterais para os animais, tornou-se uma preocupação. Vários estudos têm demonstrado interação positiva com extratos de plantas e fármacos, que permitem a redução da concentração necessária para produzir o efeito desejado, minimizando os efeitos adversos. Este estudo determinou a eficiência da combinação da fração diclorometânica (DCM) de Ageratum conyzoides L. com atividade anti-Ehrlichia canis associada com doxiciclina por meio do ensaio de Checkerboard. O material vegetal foi coletado em São Luís, Maranhão, nordeste do Brasil, seguido pela extração em MeOH:H2O (8:2) e partição da fração diclorometânica. Após a determinação da Concentração Inibitória Mínima (CIM) da fração em estudo frente às células DH82 infectadas com Ehrlichia canis, a mesma foi combinada com a doxiciclina para derivação do Índice de Concentração Inibitória da Fração (Índice CIF). Observou-se uma redução de 5,83 vezes a concentração inibitória mínima da doxiciclina mostrando que esta fração de A. conyzoides, composta predominantemente por lignanas identificadas por espectrometria de massas, notavelmente intensificou a atividade desse fármaco contra E. canis, resultando em um efeito sinérgico.

Cytotoxicity and Pro-Apoptotic, Antioxidant and Anti-Inflammatory Activities of Geopropolis Produced by the Stingless Bee Melipona fasciculata Smith.

Geopropolis is produced by some stingless bee species, such as Melipona fasciculata Smith, a native species from Brazil. This study aims to investigate the antioxidant and anti-inflammatory activities and cytotoxicity effects of geopropolis hydroethanolic extracts against lung (H460 and A549) and ovarian (A2780 and ES2) cancer cell lines and non-tumor (HUVEC) cell lines using chemical identification by LC/MS/MS analysis and in silico assays to determine which compounds are associated with bioactivity. The antioxidant activity of extracts and inhibitory activity against COX enzymes were assessed by in vitro assays; cytotoxicity effect was evaluated by the MTT assay; cell cycle was assessed by flow cytometry and apoptosis by Western blotting. The geopropolis extracts showed great radical scavenging potential, preferential inhibition of COX-2, decreased cancer cell viability, non-cytotoxic effects against the non-tumoral cell line, besides modulating the cell cycle and inducing cancer cell apoptosis through the activation of caspase-3 and PARP protein cleavage. The in silico study suggests that corilagin, typhaneoside, taraxerone and marsformosanone, identified by LC/MS/MS, can be associated with anti-inflammatory activity and cytotoxic effects. Thus, the current study suggests the potential of geopropolis concerning the research field of new pharmacological alternatives regarding cancer therapy.

Influence of the Phenological State of in the Antioxidant Potential and Chemical Composition of Ageratina havanensis. Effects on the P-Glycoprotein Function.

Ageratina havanensis (Kunth) R. M. King & H. Robinson is a species of flowering shrub in the family Asteraceae, native to the Caribbean and Texas. The aim of this work was to compare the quantitative chemical composition of extracts obtained from Ageratina havanensis in its flowering and vegetative stages with the antioxidant potential and to determine the effects on P-glycoprotein (P-gp) function. The quantitative chemical composition of the extracts was determined quantifying their major flavonoids by UPLC-ESI-MS/MS and by PCA analysis. The effects of the extracts on P-gp activity was evaluated by Rhodamine 123 assay; antioxidant properties were determined by DPPH, FRAP and inhibition of lipid peroxidation methods. The obtained results show that major flavonoids were present in higher concentrations in vegetative stage than flowering stage. In particular, the extracts obtained in the flowering season showed a significantly higher ability to sequester free radicals compared to those of the vegetative season, meanwhile, the extracts obtained during the vegetative stage showed a significant inhibitory effect against brain lipid peroxidation and a strong reductive capacity. This study also showed the inhibitory effects of all ethanolic extracts on P-gp function in 4T1 cell line; these effects were unrelated to the phenological stage. This work shows, therefore, the first evidence on: the inhibition of P-gp function, the antioxidant effects and the content of major flavonoids of Ageratina havanensis. According to the obtained results, the species Ageratina havanensis (Kunth) R. M. King & H. Robinson could be a source of new potential inhibitors of drug efflux mediated by P-gp. A special focus on all these aspects must be taking into account for future studies.

Anti-Inflammatory and Antioxidant Activity of Pollen Extract Collected by Scaptotrigona affinis postica: in silico, in vitro, and in vivo Studies.

Bees are of great importance for plant diversity for being an important pollinating agents. Stingless bees such as Scaptotrigona affinis postica, is cultivated largely due to the products offered by it. Pollen is one of these products, which has been highlighted for exhibit various therapeutic properties. Considering the bioactivity of this natural product, this study investigated the antioxidant, anti-inflammatory, antinociceptive activities, and elucidated the chemical composition of pollen collected extract by Scaptotrigona affinis postica. Using in vitro assays, the antioxidant potential and inhibitory activity against the COX enzyme from pollen extract was evaluated. Additionally, tests were performed to measure the anti-inflammatory and antinociceptive activities in animal models. In our results, we found that pollen extract showed antioxidant effects and inhibitory activity against the COX enzyme. The in vivo assays showed that the extract acts on the nervous system in local and systemic levels and that the anti-inflammatory activity is due the prostanoids reducing. Chemical analyses recognize 10 molecules in the extract belonging to the polyphenol and flavonoids classes and the computational study suggests that is responsible for the observed results. Thus, it is reported for the first time the biological potential of S. aff. postica pollen extract and we conclude that this bee product can be considered as one source of potential new drugs.

Anti-Inflammatory and Antinociceptive Activity of Pollen Extract Collected by Stingless Bee Melipona fasciculata.

The stingless bee, Melipona fasciculata Smith (Apidae, Meliponini), is a native species from Brazil. Their products have high biotechnological potential, however there are no studies about the biological activities of pollen collected by M. fasciculata. In this context, the present study investigated the chemical composition, anti-oxidant, anti-inflammatory, and analgesic activities of hydroethanolic pollen extracts collected by M. fasciculata in three cities in Maranhão State, Brazil. We verified the antioxidant activity of the extracts and inhibitory activity against the cyclooxygenase enzyme using in vitro assays and in allowed to select the extract with higher efficiency to be used on in vivo assays. In these trials, the selected extract showed high anti-inflammatory activity as well as nociceptive effects at central and peripheral level, suggesting that this extract acts on inhibition of histamine release and decreased synthesis of prostaglandins and the in-silico study suggested that polyphenols and acids fatty acids in the extract may be associated with these activities. The results of the present study report the high biological potential of pollen extract and we conclude that the pollen collected by M. fasciculata can be considered as the object of research for new pharmacological alternatives.

Antioxidant and hepatoprotective effects of ethanolic and ethyl acetate stem bark extracts of Copaifera multijuga (Fabaceae) in mice / Efeitos antioxidante e hepatoprotetor dos extratos brutos etanólico e acetato de etila da casca do caule da Copaifera multijuga(Fabaceae) em camundongos

The properties of oil-resin of copaiba, Copaifera multijuga are commonly mentioned in the literature, but there are few studies on extracts from its stem bark. We evaluated the antioxidant effects of ethanolic (EE) and ethyl acetate (EA) crude stem bark extracts from copaiba and compared them to rutin in a paracetamol (PCM)-induced oxidative stress model in mice. All test comparisons differed significantly. Hepatic catalase (CAT) and glutathione-S-transferase (GST) activity decreased in the PCM group, and there was an increase of protein carbonyls in the liver, kidney and brain. However, the protein carbonyls decreased in the liver for the PCM + EE group, in the kidneys for the PCM + EA and PCM + Rutin groups, and in the brain for all treatments. Hepatic GSH decreased in the PCM group and increased in the PCM + EE group. The extracts showed a positive effect on ascorbic acid (ASA), since they were able to restore the levels of parameters that had been changed by PCM. There was an increase of ALT and AST activity in the plasma within the PCM group. Even though ALT decreased in the PCM + Rutin, PCM + EE and PCM + EA groups, EE and EA did not have an effect on AST. The strongest antioxidant effect was observed for EE, due to the presence of the phenolic compounds epicatechin and epiafzelechin, as well as the highest concentration of total phenols and an excellent antioxidant potential observed in the DPPH· test.

As propriedades do óleo-resina da copaíba, Copaifera multijuga são comumente citadas na literatura, mas há poucos estudos sobre extratos da casca do caule. Avaliamos os efeitos antioxidantes de extratos brutos etanólico (EE) e acetato de etila (EA) da casca do caule da copaíba e os comparamos à rutina no modelo de estresse oxidativo induzido por paracetamol (PCM) em camundongos. Todas as comparações de teste diferiram significativamente. A atividade da catalase hepática (CAT) e da glutationa-S-transferase (GST) diminuiu no grupo PCM, e houve um aumento de proteínas carboniladas no fígado, rim e cérebro. No entanto, as proteínas carboniladas diminuíram no fígado para o grupo PCM + EE, nos rins para os grupos PCM + EA e PCM + rutina, e no cérebro para todos os tratamentos. A GSH hepática diminuiu no grupo PCM e aumentou no grupo PCM + EE. Os extratos mostraram um efeito positivo sobre o ácido ascórbico (ASA), uma vez que foram capazes de restaurar os níveis dos parâmetros que foram alterados pelo PCM. Houve um aumento da atividade de ALT e AST no plasma dentro do grupo PCM. Embora a ALT tenha diminuído nos grupos PCM + rutina, PCM + EE e PCM + EA, EE e EA não afetaram a AST. O efeito antioxidante mais forte foi observado para o EE, provavelmente devido à presença dos compostos fenólicos epicatequina e epiafzelequina, assim como à maior concentração de fenóis totais e um excelente potencial antioxidante observado no teste DPPH·