Synthesis and Evaluation of New Phytotoxic Fluorinated Chalcones as Photosystem II and Seedling Growth Inhibitors.

The development of novel phytotoxic compounds has been an important aim of weed control research. In this study, we synthesized fluorinated chalcone derivatives featuring both electron-donating and electron-withdrawing groups. These compounds were evaluated both as inhibitors of the photosystem II (PSII) electron chain as well as inhibitors of the germination and seedling growth of Amaranthus plants. Chlorophyll a (Chl a) fluorescence assay was employed to evaluate their effects on PSII, while germination experiments were conducted to assess their impact on germination and seedling development. The results revealed promising herbicidal activity for (E)-3-(4-bromophenyl)-1-(4-fluorophenyl)prop-2-en-1-one (7 a) and (E)-1-(4-fluorophenyl)-3-phenylprop-2-en-1-one (7 e). Compounds 7 a and 7 e exhibited a reduction in Chl a parameters associated with performance indexes and electron transport per reaction center. This reduction suggests a decrease in PSII activity, attributed to the blockage of electron flow at the quinone pool. Molecular docking analyses of chalcone derivatives with the D1 protein of PSII revealed a stable binding conformation, wherein the carbonyl and fluorine groups interacted with Phe265 and His215 residues, respectively. Additionally, at a concentration of 100 µM, compound 7 e demonstrated pre- and post-emergent herbicidal activity, resulting in a reduction of the seed germination index, radicle and hypocotyl lengths of Amaranthus weeds.

Inhibition of mycelial growth, conidial germination, and Botrytis cinerea Pers: Fr colonization in begonia with biocompatible products

This study evaluated the effects of potassium and sodium carbonate and bicarbonate, Bacillus subtilis (Cohn, 1872) QST-713, Bacillus pumilus (Meyer & Gottheil, 1901) QST-2808, and crude and roasted coffee oils on the inhibition of mycelial growth and conidial germination in Botrytis cinerea Pers.: Fr and the colonization of begonia (Begonia elatior Hort. ex Steud) leaf discs by B. cinerea inoculated before, simultaneously and after with these alternative products. The assays were carried out using the Baladin begonia cultivar. The inhibition of B. cinerea mycelial growth and conidial germination was proportional to increases in the concentration of all the products. The inhibition of conidial germination was directly proportional to the concentrations of B. pumilus QST-2808 and B. subtilis QST-713. Coffee oils were less efficient in inhibiting germination than the other products. The crude and roasted coffee oils, potassium and sodium carbonates and bicarbonates, and B. pumilus and B. subtilis sprayed 24 h before, simultaneously, or 24 h after pathogen inoculation inhibited the colonization of begonia leaf discs by B. cinerea. The positive results for the suppression of B. cinerea by the alternative products tested herein merit scrutiny. There is a pressing need to evaluate these products in the management of gray mold, as the severity of this disease is usually high under favorable conditions in greenhouses.

Identification of Potential Insect Growth Inhibitor against Aedes aegypti: A Bioinformatics Approach.

Aedes aegypti is the main vector that transmits viral diseases such as dengue, hemorrhagic dengue, urban yellow fever, zika, and chikungunya. Worldwide, many cases of dengue have been reported in recent years, showing significant growth. The best way to manage diseases transmitted by Aedes aegypti is to control the vector with insecticides, which have already been shown to be toxic to humans; moreover, insects have developed resistance. Thus, the development of new insecticides is considered an emergency. One way to achieve this goal is to apply computational methods based on ligands and target information. In this study, sixteen compounds with acceptable insecticidal activities, with 100% larvicidal activity at low concentrations (2.0 to 0.001 mg·L−1), were selected from the literature. These compounds were used to build up and validate pharmacophore models. Pharmacophore model 6 (AUC = 0.78; BEDROC = 0.6) was used to filter 4793 compounds from the subset of lead-like compounds from the ZINC database; 4142 compounds (dG < 0 kcal/mol) were then aligned to the active site of the juvenile hormone receptor Aedes aegypti (PDB: 5V13), 2240 compounds (LE < −0.40 kcal/mol) were prioritized for molecular docking from the construction of a chitin deacetylase model of Aedes aegypti by the homology modeling of the Bombyx mori species (PDB: 5ZNT), which aligned 1959 compounds (dG < 0 kcal/mol), and 20 compounds (LE < −0.4 kcal/mol) were predicted for pharmacokinetic and toxicological prediction in silico (Preadmet, SwissADMET, and eMolTox programs). Finally, the theoretical routes of compounds M01, M02, M03, M04, and M05 were proposed. Compounds M01−M05 were selected, showing significant differences in pharmacokinetic and toxicological parameters in relation to positive controls and interaction with catalytic residues among key protein sites reported in the literature. For this reason, the molecules investigated here are dual inhibitors of the enzymes chitin synthase and juvenile hormonal protein from insects and humans, characterizing them as potential insecticides against the Aedes aegypti mosquito.

Imunoexpressão da proteína ING3 em Queilites Actínicas e carcinomas de células escamosas de lábio inferior / Immunoexpression of ING3 protein in actinic cheilitis and squamous cell carcinomas of the lower lip

A família de inibidores de crescimento (inhibitor of growth - ING) corresponde a um grupo de genes supressores tumorais cuja expressão se apresenta alterada ou ausente em diversos tipos de câncer. Os produtos destes genes estão relacionados, principalmente, a processos celulares indispensáveis na carcinogênese, como remodelação da cromatina, proliferação celular, replicação e reparo do DNA. Este estudo avaliou a expressão imuno-histoquímica da proteína ING3 em 45 espécimes de queilite actínica (QA) e 48 espécimes de carcinoma de células escamosas de lábio inferior (CCELI). A expressão da proteína foi comparada entre os dois grupos de amostras, bem como com os parâmetros clínico-patológicos das lesões estudadas, através dos testes estatísticos Exato de Fisher, Kruskal-Wallis (KW), Mann-Whitney (U) e teste de correlação de Spearman. Um nível de significância de 5% foi adotado para todos os testes, sendo considerados significativos os valores de p ≤ 0,05. Não foram encontradas associações estatisticamente significativas entre as variáveis morfológicas de CCELI e tamanho do tumor, envolvimento linfonodal, estadiamento clínico, recorrência local e metástase linfonodal após tratamento (p>0,05). Óbitos foram significativamente mais frequentes em tumores de alto escore de risco histopatológico (p<0,05). Nas QAs, diferenças significativas na expressão de ING3 núcleo-citoplasmática, e restrita ao citoplasma, com a gradação de Kujan et al. (2006) foram encontradas (p<0,05). Nos CCELIs, não houve diferença estatisticamente significativa ao comparar as expressões de ING3 (núcleo-citoplasmática e restrita ao citoplasma) com os parâmetros clínicos e morfológicos (p>0,05). A expressão de ING3 núcleo-citoplasmática foi significativamente menor em CCELI quando comparada aos casos de QA (p<0,05) e a expressão restrita ao citoplasma foi significativamente maior nos CCELIs (p<0,05). Nossos resultados sugerem que há notável diminuição da expressão nuclear de ING3 conforme a progressão maligna, indicando função supressora tumoral prejudicada desta proteína em QAs e CCELIs. Entretanto, acredita-se que, na carcinogênese labial, ING3 caracteriza-se melhor como um marcador preditor de transformação maligna, do que um biomarcador de comportamento biológico tumoral (AU).

The family of inhibitors of growth (ING) corresponds to a group of tumor suppressor genes whose expression is altered or absent in several types of cancer. The products of these genes are mainly related to cellular processes indispensable in carcinogenesis, including cell proliferation, DNA replication and repair. This study evaluated the immunohistochemical expression of ING3 protein in 45 actinic cheilitis (AC) and 48 lower lip squamous cell carcinoma (CCELI) specimens. Protein expression was compared between the two groups of samples, as well as with the clinicopathological parameters of studied lesions, using Fisher's exact tests, Kruskal-Wallis (KW), Mann-Whitney (U) and correlation test of Spearman. A significance level of 5% was adopted for all tests, with values of p ≤ 0.05 being considered significant. No statistically significant associations were found between morphological variables of CCELI and tumor size, lymph node involvement, clinical staging, local recurrence and lymph node metastasis after treatment (p>0.05). Deaths were significantly more frequent in tumors with a high histopathological risk score (p<0.05). In ACs, significant differences in nuclear-cytoplasmic and restricted to the cytoplasm expression of ING3, with gradation of Kujan et al. (2006) were found (p<0.05). In CCELIs, there was no statistically significant difference when comparing ING3 expressions (nucleocytoplasmic and cytoplasmic restricted) with clinical and morphological parameters (p>0.05). Nucleocytoplasmic ING3 expression was significantly lower in CCELI when compared to AC cases (p<0.05) and cytoplasm-restricted expression was significantly higher in CCELIs (p<0.05). Our results suggest that there is a remarkable decrease in ING3 nuclear expression according to malignant progression, indicating an impaired tumor suppressor function of this protein in AC and CCELI. However, it is believed that, in lip carcinogenesis, ING3 is better characterized as predictive marker of malignant transformation, rather than a biomarker of tumor biological behavior (AU).

Evaluation of Chalcone Derivatives as Photosynthesis and Plant Growth Inhibitors.

We report the evaluation of chalcone derivatives as photosystem II (PSII) and plant growth inhibitors. Chalcone derivatives were evaluated as PSII inhibitors through Chl a fluorescence measurement. (E)-Chalcone (6a) and (E)-3-(4-bromophenyl)-1-(4-fluorophenyl)prop-2-en-1-one (6j) showed the best results, reducing the performance index on absorption basis parameter (PIabs ) by 70 %. Additionally, the decrease of TR0 /RC and ET0 /RC parameters indicates that the chalcone derivatives limited the number of active PSII reaction centers and the amount of trapped energy within them. Compounds 6a and 6j both act as post-emergent herbicides at 50 µM, reducing the root biomass of the Ipomoea grandifolia weed by 72 % and 83 %, respectively, corroborating the fluorescence results. The selectivity against weeds as compared to valuable crops by compounds 6a and 6j were evaluated employing Zea mays and Phaseolus vulgaris plants. In these, our newly synthesized compounds showed no effects on biomass accumulation of roots and aerial parts when compared to the control, providing valuable evidence for the role of these compounds as selective inhibitors of the growth of undesired weeds.

Amazon Fruits Inhibit Growth and Promote Pro-apoptotic Effects on Human Ovarian Carcinoma Cell Lines.

Murici (Byrsonima crassifolia (L.) Kunth and B. verbascifolia (L.) DC.) and tapereba (Spondias mombin) are Amazonian fruits that contain bioactive compounds. Biochemical and molecular characterization of these fruits can reveal their potential use in preventing diseases, including cancer. The extracts were characterized regarding the presence and profile of carotenoids by high-performance liquid chromatography (HPLC), total phenolic content by the Folin-Ciocalteu assay, and antioxidant activity by antioxidant value 2,2-diphenyl-1-picrylhydrazyl (DPPH) content analysis, 22,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) content analysis, Ferric-Reducing Ability of Plasma (FRAP), and Oxygen Radical Absorbance Capacity (ORAC) analysis. The extracts of tapereba and murici studied were important sources of total carotenoids and lutein, respectively. The extracts were then tested for their effect on the viability of the A2780 ovarian cancer (OC) cell line and its cisplatin (CDDP)-resistant derived cell line, called ACRP, by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Their influence on cell cycle and apoptosis were analyzed by using flow cytometry. Murici and tapereba cell extracts exhibited a strong bioactivity by inhibiting A2780 and ACRP cell viability by 76.37% and 78.37%, respectively, besides modulating the cell cycle and inducing apoptotic cell death. Our results open new perspectives for the development of innovative therapeutic strategies using these Amazon fruit extracts to sensitize ovarian cancer cells to current chemotherapeutic options.

Interaction of 4-ethylphenol, pH, sucrose and ethanol on the growth and fermentation capacity of the industrial strain of Saccharomyces cerevisiae PE-2.

Volatile phenols such as 4-ethylphenol are produced from hydroxycinnamic acids by Dekkera bruxellensis, an important yeast contaminating alcoholic fermentations. 4-ethylphenol results from the decarboxylation and reduction of p-coumaric acid, a compound found in sugarcane musts. In wine, volatile phenols are responsible by sensorial alterations whereas in the context of bioethanol fermentation, little is known about their effects on the main yeast, Saccharomyces cerevisiae. Here we evaluated the interaction of 4-ethylphenol and pH, sucrose and ethanol on the growth and fermentation capacity of the industrial strain of S. cerevisiae PE-2. A central compound rotational design was utilized to evaluate the effect of 4-ethylphenol, pH, ethanol and sucrose concentration on the yeast maximum specific growth rate (µmax) in microplate experiments in YPS medium (Yeast extract-Peptone-Sucrose), at 30 °C. Following, single-cycle fermentations in YPS medium, pH 4.5, 17% sucrose, at 30 °C, with 4-ethylphenol in concentrations of 10 and 20 mg L-1 being added at the start or after 4 h of fermentation, were carried out. 4-ethylphenol affected µmax of S. cerevisiae in situations that resemble the conditions of industrial bioethanol production, especially the low pH of the fermentation medium and the high ethanol concentration because of the anaerobic sucrose uptake. The addition of 4-ethylphenol on fermentation resulted in significant effect on the cell yeast concentration, pH and alcohol production, with significant decrease from 86% to the range of 65-74% in the fermentative efficiency. The industrial yeast S. cerevisiae PE-2 growth and fermentative capacity were affected by the presence of 4-ethylphenol, a metabolite produced by D. bruxellensis, which may contribute to explain the impact of this yeast on bioethanol industrial production.

Production, structural characterization, and antiproliferative activity of exopolysaccharide produced by Scleroderma areolatum Ehrenb with different carbon source.

The effects of different three carbon sources, that is, glucose, fructose, and sucrose, on production, molecular properties and antiproliferative activity of exopolysaccharide (EPS), were evaluated in the submerged culture of Scleroderma areolatum Ehrenb. Among carbon sources examined, the addition of sucrose maximizes the mycelia production, while fructose could maximize the EPS yield. Although the predominant carbohydrate compositions identified were gluconic acid and mannose, the monosaccharide composition of EPSs was also different significantly. FT-IR spectral analysis revealed there was no significant difference among the prominent characteristic groups in three EPSs. The molecular weight of EPSs was also affected by carbon source, being generally lower compared with that with glucose. However, all EPSs molecule existed as nearly globular shape form in aqueous solution. The variation of carbon sources also affected antiproliferative activity examined in vitro using cell proliferation assay. Fructose was optimal carbon source giving higher antiproliferative activity probably due to the relatively high contents of xylose in the EPS with low molecular weight.

Synthesis and evaluation of indole derivatives as photosynthesis and plant growth inhibitors.

Indole derivatives were synthetized based on the Fischer indole methodology using different phenyl hydrazine hydrochlorides and either cyclohexanone or 2-butanone. The pre- and post-emergent herbicidal activities were evaluated against Ipomoea grandifolia. A carbazole, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole (3b), decreased the PIabs parameter by 32% and increased the cross-section related parameters, indicating the inactivation of the reaction center on photosystem II. Compound 3b acts as a post-emergent herbicide prototype since dry biomass was reduced by 50%, corroborating the fluorescence results. Comparing instead with a germination experiment, 2,3,4,9-tetrahydro-1H-carbazole (3a) was found to be the most effective agent, inhibiting seed germination by 22% and decreasing root length by 50%. The tetrahydrocarbazoles showed better results than indole derivatives potentially due to the presence of methylene groups at structures, which increase the compounds' lipophilicity and may facilitate their access to the plant. In addition, electron withdrawing groups on the aromatic ring were found to correlate with increased herbicide activity. Further optimization of this series towards the development of herbicides is ongoing.

Expressão imuno-histoquímica de proteínas ING em desordens potencialmente malignas / Immunohistochemical expression of ING proteins in potentially malignant disorders

As desordens potencialmente malignas (DPM) são lesões que podem acometer a cavidade oral e que apresentam um potencial de transformação maligna. Biomarcadores moleculares têm sido estudados com o objetivo de auxiliar na predição do risco de transformação maligna dessas lesões. Dentre eles, destacam-se as proteínas ING (1-5) (do inglês, inhibitor of growth). O objetivo deste trabalho é avaliar a expressão das proteínas ING-1 e ING-2 em lesões diagnosticadas como hiperceratose e displasia epitelial (DE) (leve, moderada e severa), e correlacionar o padrão de expressão observado com o grau de displasia epitelial. Trata-se de um estudo transversal retrospectivo, de caráter semi-quantitativo e comparativo. A amostra consistiu de 60 espécimes de hiperceratose e DE, os quais foram avaliados morfologicamente e reclassificados de acordo com o sistema binário de classificação das DE proposto por Kujan et al. (2006). A imunoexpressão para ING-1 e ING-2 nas lesões estudadas foi avaliada de forma semi-quantitativa a partir da atribuição de escores, que variavam de 0 a 4, de acordo com o percentual de células epiteliais positivas para aqueles marcadores. Para a análise estatística, foram realizados os testes de Mann-Whitney e de Spearman (p ≤ 0,05). Dos 60 casos analisados, 37 (61,7%) apresentaram-se como lesões de baixo risco e 23 (38,3%) como de alto risco. Tanto para ING-1 quanto para ING-2, 93,3% das lesões displásicas estudadas apresentaram marcação citoplasmática e nuclear nas células epiteliais, com predominância de positividade nas camadas basal e suprabasal. Em relação aos escores de marcação imunohistoquímica, houve predominância de casos com elevada expressão (escore 4) para ambos os marcadores. No entanto, ao se comparar estes escores (nuclear, citoplasmática e geral) com a gradação histológica das DE, tanto para ING-1 quanto para ING-2, não foi observada diferença estatisticamente significativa. Dessa forma, os resultados do presente estudo sugerem que a imunoexpressão de ING-1 e ING-2 não têm relação com o grau de DE oral. No entanto, a alta expressão observada nessas lesões sugere que estas proteínas estariam envolvidas no processo da carcinogênese oral (AU).

Potentially malignant disorders (PMD) are lesions that can affect the oral cavity and present a potential for malignant transformation. Molecular biomarkers have been studied to help predict the risk of malignant transformation of these lesions. Among them, the ING (inhibitor of growth) proteins (1-5) are highlighted. The objective of this study is to evaluate the expression of ING-1 and ING-2 proteins in lesions diagnosed as hyperkeratosis and ED (mild, moderate and severe), and to correlate the expression pattern observed with the degree of epithelial dysplasia. This is a cross-sectional, retrospective, semi-quantitative and comparative study. The sample consisted of 60 specimens of hyperkeratosis and epithelial dysplasia (ED), which were morphologically evaluated and reclassified according to the binary classification system of ED proposed by Kujan et al. (2006). The immunoexpression for ING-1 and ING-2 in the studied lesions was evaluated semi-quantitatively from the assignment of scores ranging from 0 to 4, according to the percentage of epithelial cells positive for those markers. For the statistical analysis, the Mann-Whitney and Spearman tests (p ≤ 0.05) were performed. Of the 60 cases analyzed, 37 (61.7%) presented as low-risk lesions and 23 (38.3%) as highrisk lesions. For both ING-1 and ING-2, 93.3% of the dysplastic lesions studied presented cytoplasmic and nuclear marking on epithelial cells, with predominance of positivity in the basal and suprabasal layers. Regarding the immunohistochemical marking scores, there was a predominance of cases with high expression (score 4) for both markers. However, when comparing these scores (nuclear, cytoplasmic and general) with the histological gradation of ED, for both ING-1 and ING-2, no statistically significant difference was observed. Thus, the results of the present study suggest that immunoexpression of ING-1 and ING-2 is not related to the degree of oral ED. However, the high expression observed in these lesions suggests that these proteins would be involved in the oral carcinogenesis process (AU).

Antiproliferative Activity of Walnut (Juglans regia L.) Proteins and Walnut Protein Hydrolysates.

Proteins from Juglans regia L. were isolated. Then, proteins were hydrolyzed with different enzymes. Antiproliferative activity of proteins and of the protein hydrolysates of J. regia L. were evaluated using the sulforhodamine B method. Glutelin and prolamin proteins presented a high antiproliferative activity against cancer cells PC-3 (prostate) and K-562 (leukemia) with values of 43.9 and 84.4 µg/mL, respectively. The highest inhibitory effect observed was 50% at 0.25 µg/mL concentration in gastrointestinal digestion with pepsin and corolase pp in a dose-dependent manner against cancer cell UACC62 (melanoma). Pepsin hydrolysate showed inhibitory effects against cancer cell UACC62 (melanoma) with a concentration of 71.0 µg/mL. The effects were studied in a dose-dependent manner. The hydrolysate obtained with neutrase enzyme presented inhibitory effects against cancer cell UACC62 (melanoma) at a concentration of 25 µg/mL. Neither proteins nor protein hydrolysates presented cytotoxicity against normal cell assay VERO (epithelial).

Risk Assessment via Metabolism and Cell Growth Inhibition in a HepG2/C3A Cell Line Upon Treatment with Arpadol and its Active Component Harpagoside.

Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetic/pharmacodynamic modeling of etoposide tumor growth inhibitory effect in Walker-256 tumor-bearing rat model using free intratumoral drug concentrations.

The purpose of this study was to establish a population pharmacokinetic/pharmacodynamic (PK/PD) model linking etoposide free tumor and total plasma concentrations to the inhibition of solid tumor growth in rats. Walker-256 tumor cells were inoculated subcutaneously in the right flank of Wistar rats, which were randomly divided in control and two treated groups that received etoposide 5 or 10mg/kg i.v. bolus every day for 8 and 4days, respectively, and tumor volume was monitored daily for 30days. The plasma and intratumoral concentrations-time profiles were obtained from a previous study and were modeled by a four-compartment population pharmacokinetic (popPK) model. PK/PD analysis was conducted using MONOLIX v.4.3.3 on average data and by mean of a nonlinear mixed-effect model. PK/PD data were analyzed using a modification of Simeoni Tumor Growth Inhibition (TGI) model by introduction of an Emax function to take into account the concentration dependency of k2variable parameter (variable potency). The Simeoni TGI-Emax model was capable to fit schedule-dependent antitumor effects using the tumor growth curves from the control and two different administered schedules. The PK/PD model was capable of describing the tumor growth inhibition using total plasma or free tumor concentrations, resulting in higher k2max (maximal potency) for free concentrations (25.8mL·µg-1·day-1 - intratumoral vs. 12.6mL·µg-1·day-1 total plasma). These findings indicate that the plasma concentration may not be a good surrogate for pharmacologically active free tumor concentrations, emphasizing the importance of knowing drug tumor penetration to choose the best antitumor therapy.

Apoptotic effect of chalcone derivatives of 2-acetylthiophene in human breast cancer cells.

BACKGROUND: A variety of chalcones have demonstrated cytotoxic activity toward several cancer cell lines. This study aimed to investigate the cytotoxicity of four chalcones derivatives of 2-acetylthiophene in human breast cancer cell lines. METHODS: MCF-7 and MDA-MB-231 cells were treated with synthesized chalcones and the cytotoxicity was evaluated by tetrazolium dye (MTT), live/dead, and DAPI assays. RESULTS: Chalcones significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner. After 48h treatment, the IC50 values ranging from 5.52 to 34.23µM. Chalcone 3c displayed the highest cytotoxic activity from all the tested compounds. Cytotoxic effects of compounds were confirmed in the live/dead assay. In addition, DAPI staining revealed that these compounds induce death by apoptosis. CONCLUSION: The data speculate that chalcone derivatives of 2-acetylthiophene may represent a source of therapeutic agents for human breast cancer.

Estudos estruturais e funcionais de Tsa1 de Saccharomyces cerevisiae: um modelo biológico para o estudo de inibidores de crescimento celular para leucemia linfoide aguda / Functional and structural evaluation of Tsa1 from Saccharomyces cerevisiae: a biological model for study of cellular growth inhibitors for acute lymphocytic leukemia

As peroxirredoxinas (Prx) são enzimas antioxidantes que se destacam pela capacidade de decompor uma grande variedade de hidroperóxidos com elevada eficiência (106-108M-1s-1), mantendo essas moléculas em níveis adequados à homeostase celular. Entretanto, já foi demonstrado que em diversos tipos tumorais os níveis de Prx são extremamente aumentados e experimentos envolvendo sua inativação resultam na diferenciação ou apoptose de células tumorais. Recentemente, foi descoberto um diterpenóide denominado adenantina que seria o primeiro inibidor para as Prx1 e Prx2 de humanos e foi demonstrada que sua aplicação em células de leucemia mieloide aguda promoveu diferenciação ou apoptose dessas células. Nesse contexto, o presente trabalho apresenta duas vertentes: 1) A caracterização das alterações estruturais e funcionais promovidas pela ligação da adenantina ao sítio ativo das Prx utilizando Tsa1 de Saccharomyces cerevisiae como modelo biológico, em função da sua alta similaridade com Prx2 de humanos; 2) Avaliação da atividade antitumoral dose dependente de adenantina sobre as linhagens celulares REH e MOLT-4 de leucemia linfoide aguda. No que concerne à primeira linha de investigação, nossos resultados revelam que Tsa1 é suscetível à inibição por adenantina, uma vez que o tratamento reduziu em ~66 % a velocidade de decomposição de peróxido de hidrogênio. Adicionalmente, a mutação da Thr44 de Tsa1, pertencente à chamada tríade catalítica, por uma Ser resultou em uma proteína mais suscetível a alterações na estrutura secundária e à inibição da atividade peroxidásica em função da ligação com adenantina, apresentando uma diminuição de ~85% na velocidade de reação. Características semelhantes foram observadas para a proteoforma Tsa2 de S. cerevisiae, que carreia naturalmente a substituição da Thr44 pela Ser. Análises de sequências de Prx em bancos de dados revelaram que majoritariamente proteínas contendo Ser são encontradas em organismos procariotos, muitos deles patogênicos. Finalmente, demonstramos por meio de ensaios citotoxicidade que as bactérias Staphylococcus aureus e Staphylococcus epidermidis, que possuem uma Ser na tríade catalítica, têm seu crescimento inibido pelo tratamento com adenantina (IC50 de 460µM e 77µM, respectivamente), enquanto que para Escherichia coli, que possui Thr nessa posição, a toxicidade da adenantina foi bastante baixa (não foi possível determinar o IC50 nas condições utilizadas). Dessa forma, os dados apresentados neste trabalho demonstram o potencial da utilização da adenantina tanto como antibiótico quanto como antileucêmico

Peroxiredoxins (Prx) are antioxidant enzymes which stand out due the ability to decompose a wide variety of hydroperoxides with high efficiency (106-108M-1s-1) maintaining these molecules at suitable levels to cellular homeostasis and participating in several signaling events. However, it has been shown that, in many tumor types, Prx levels are extremely increased and experiments involving its inactivation have resulted in differentiation or apoptosis of tumor cells. It was recently found a diterpenoid, called adenanthin, that would be the first human Prx1 and Prx2 inhibitor and it was demonstrated that its application in acute myeloid leukemia cells was able to promote differentiation or apoptosis. In this context, this work presents two lines of research: 1) Characterization of structural and functional changes promoted by adenanthin binding to Prx active site using Tsa1 from Saccharomyces cerevisiae as biological model, due to its high similarity to human Prx2. 2) Evaluation of adenanthin dose-dependent antitumor activity over the acute lymphoid leukemia cell lines REH and MOLT-4. As regards the first line of research, our result reveal that Tsa1 is susceptible to inhibition by adenanthin, since the treatment with this binder reduced the hydrogen peroxide decomposition velocity in ~ 66%. In addition, the replacement of Thr44 from Tsa1, aminoacid belonging to the so-called catalytic triad, by a Ser resulted in a protein more susceptible to alterations in secondary structure and to peroxidase activity inhibition in function of adenanthin binding, presenting ~85% of decrease in reaction velocity. Similar characteristics were observed for Tsa2 proteoform from S. cerevisiae, which naturally carries the substitution of Thr44 by Ser. Prx sequences analyzes in databases revealed that mostly Ser-containing proteins are found in prokaryotic organisms, many of them pathogenic ones. Finally, we demonstrate through cytotoxicity assays that the bacteria Staphylococcus aureus and Staphylococcus epidermidis, which have a Ser in catalytic triad, have their growth inhibited by adenanthin treatment (IC50 of 460µM and 77µM, respectively), whereas for Escherichia Coli, which has Thr at that position, the tocyxicity of adenanthin was quite low (it was not possible to determine the IC50 under the used conditions). Regarding the second line of investigation, we found that adenanthin is able to induce the death of leukemic cell lines REH and MOLT-4, and for the last one, there was an unexpected proliferation of cells treated by the longest incubation period (72 hours), characterizing a possible indication of differentiation process. In this sense, the data presented here demonstrate the potential of adenanthin use in both antibiotic and antileukemic treatment

Copaifera langsdorffii oleoresin and its isolated compounds: antibacterial effect and antiproliferative activity in cancer cell lines.

BACKGROUND: Natural products display numerous therapeutic properties (e.g., antibacterial activity), providing the population with countless benefits. Therefore, the search for novel biologically active, naturally occurring compounds is extremely important. The present paper describes the antibacterial action of the Copaifera langsdorffii oleoresin and ten compounds isolated from this oleoresin against multiresistant bacteria; it also reports the antiproliferative activity of the Copaifera langsdorffii oleoresin and (-)-copalic acid. METHODS: MICs and MBCs were used to determine the antibacterial activity. Time-kill curve assays provided the time that was necessary for the bacteria to die. The Minimum Inhbitory Concentration of Biofilm (CIMB50) of the compounds that displayed the best results was calculated. Cytotoxicity was measured by using the XTT assay. RESULTS: The diterpene (-)-copalic acid was the most active antibacterial and afforded promising Minimum Inhibitory Concentration (MIC) values for most of the tested strains. Determination of the bactericidal kinetics against some bacteria revealed that the bactericidal effect emerged within six hours of incubation for Streptococcus pneumoniae. Concerning the antibiofilm action of this diterpene, its MICB50 was twofold larger than its CBM against S. capitis and S. pneumoniae. The XTT assay helped to evaluate the cytotoxic effect; results are expressed as IC50. The most pronounced antiproliferative effect arose in tumor cell lines treated with (-)-copalic acid; the lowest IC50 value was found for the human glioblastoma cell line. CONCLUSIONS: The diterpene (-)-copalic acid is a potential lead for the development of new selective antimicrobial agents to treat infections caused by Gram-positive multiresistant microorganisms, in both the sessile and planktonic mode. This diterpene is also a good candidate to develop anticancer drugs.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons.

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Different resistance patterns of reference and field strains of Brucella abortus.

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 µg/mL and 80 µg/mL), thionin (2.5 µg/mL and 10 µg/mL), rifampicin (200 µg/mL) and safranin O (200 µg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO 2 . Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 µg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 µg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 ( B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75-0.80 (10 µg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

α-Linolenic Fatty Acid Supplementation Decreases Tumor Growth and Cachexia Parameters in Walker 256 Tumor-Bearing Rats.

Fish oil (FO) has been shown to affect cancer cachexia, tumor mass, and immunity cell. n-3 PUFA, specifically α-linolenic fatty acid (ALA), has controversial effects. We investigated this in nontumor-bearing Wistar rats fed regular chow (C), fed regular chow and supplemented with FO or Oro Inca oil (OI), and Walker 256 tumor-bearing rats fed regular chow (W), fed regular chow and supplemented with FO (WFO) or OI (WOI). Rats were supplemented (1g/kg body weight/day) during 4 wk and then the groups tumor-bearing were inoculated with Walker 256 tumor cells suspension and 14 days later the animals were killed. WFO increased EPA fivefold and DHA 1.5-fold in the tumor tissue compared to W (P < 0.05). OI supplementation increased of threefold of ALA when compared to W (P < 0.05). Tumor mass in WFO and OI was of 2.3-fold lower, as well as tumor cell proliferation of 3.0-fold tumor tissue lipoperoxidation increased of 76.6% and cox-2 expression was 20% lower. Cachexia parameters were attenuate, blood glucose (25% higher), Triacylglycerolemia (50% lower), and plasma TNF-α (65% lower; P < 0.05) and IL-6 (62.5% lower). OI, rich in ALA, caused the same effect on cancer as those seen in FO.