Indução de apoptose e ativação de caspase por chalconas em linhagem celular de leucemia: relação entre estresse oxidativo e morte celular

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2010O câncer corresponde a um grupo de doenças que possuem em comum o descontrole na proliferação. Em alguns países o câncer já assumiu o papel de principal causa de morte. No Brasil, as leucemias estão entre as 10 principais causas de morte por câncer tanto em homens quanto em mulheres. A leucemia linfóide aguda é uma desordem maligna causada pela proliferação de progenitores linfóides B e T. As chalconas são compostos intermediários essenciais para a biossíntese de flavonóides em plantas. Esta classe de moléculas tem demonstrado uma grande variedade de efeitos farmacológicos entre eles antitumoral. O objetivo deste trabalho foi estudar a atividade antitumoral de chalconas derivadas quimicamente da 3,4-metilenodioxiacetofenona e da 2-naftilacetofenona, em células murinas de leucemia linfoblástica aguda (L1210). Para isto, foi analisada a viabilidade celular nas células tumorais bem como o mecanismo de morte envolvido. Observou-se que as chalconas denominadas R7, R13 e R15 induziram citotoxicidade dependente da concentração e do tempo, apresentando IC50 na faixa de micromolar. A fragmentação do DNA e a ativação da caspase-3 evidenciam que estas chalconas são capazes de promover morte celular por apoptose. Todas as chalconas induziram a geração de espécies reativas de oxigênio, a lipoperoxidação e um aumento na atividade das enzimas antioxidantes glutationa peroxidase, glutationa redutase e glutationa s-transferase. Todas as chalconas também induziram um aumento de glutationa total o qual corresponde principalmente a glutationa oxidada e a chalcona R15 induziu um aumento da glutationa na sua forma reduzida. Estes dados sugerem que todas as chalconas induziram um estresse oxidativo nas células sendo que as chalconas R7 e R15 apresentam um maior efeito pró-oxidante podendo ser esse o principal mecanismo de morte envolvido na citotoxicidade destas moléculas. Todas as chalconas testadas também induziram depleção de ATP nas células, no entanto a chalcona R13 causou uma maior depleção em um tempo menor de incubação. Portanto, o principal mecanismo de morte envolvido com a chalcona R13 pode estar relacionado a um dano mitocondrial e conseqüente depleção energética. Considerando a citotoxicidade das chalconas testadas, as mesmas são promissoras para a continuidade das investigações

Mecanismos de citotoxicidade de chalconas isoladas e encapsuladas em nanopartículas lipídicas sobre uma linhagem celular de leucemia linfoblástica aguda e identificação de novos inibidores da proteína de resistência ABCG2

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2013.O câncer corresponde a um grupo de doenças que possuem em comum o descontrole na proliferação celular. A leucemia linfóide aguda (LLA) é uma desordem maligna causada pela proliferação de progenitores linfóides B e T e possui uma incidência maior em crianças. As chalconas são compostos intermediários essenciais para a biossíntese de flavonóides e têm demonstrado uma grande variedade de efeitos farmacológicos entre eles o efeito antitumoral. A primeira parte deste trabalho consistiu na avaliação da atividade antitumoral de três chalconas derivadas quimicamente da 2-naftilacetofenona denominadas R7, R13 e R15, em células murinas de LLA (L1210). Observou-se que as chalconas induziram apoptose nas células L1210 por diferentes mecanismos. Particularmente, as chalconas R7 e R13 induziram apoptose através das vias intrínseca e extrínseca e também como resultado de um estresse de retículo endoplasmático. A chalcona R15 induziu a apoptose através da via extrínseca e estresse de retículo endoplasmático não induzindo danos mitocondriais. Na segunda parte deste trabalho buscou-se avaliar o efeito das três chalconas encapsuladas em um sistema nanoestruturado lipídico. Um dos objetivos da encapsulação é a vetorização dos fármacos para as células tumorais melhorando a atividade antitumoral e diminuindo os efeitos adversos. Inicialmente, avaliou-se o efeito de três nanopartículas lipídicas em células não-tumorais VERO e in vivo utilizando camundongos, a fim de escolher o sistema mais biocompatível. A NE foi o único sistema que não induziu toxicidade in vitro e foi escolhida como veículo para incorporação das moléculas. As chalconas R7 e R15 encapsuladas induziram a mesma toxicidade que as moléculas livres em células de leucemia L1210. Já a encapsulação da chalcona R13 diminuiu o seu efeito antileucêmico indicando que esta molécula pode não estar sendo liberada da nanopartícula. A toxicidade das chalconas livres e encapsuladas foi avaliada em células não-tumorais (in vitro) e in vivo. Com esta avaliação, verificou-se que o encapsulamento impediu a toxicidade das chalconas em células não-tumorais VERO e in vivo, principalmente diminuindo a inflamação hepática induzida nos animais tratados com as chalconas livres. Os resultados portanto demonstraram que o encapsulamento não melhorou a atividade antileucêmica das chalconas, entretanto, diminuiu a respectiva toxicidade em células não tumorais. A terceira e última parte deste trabalho corresponde a avaliação de chalconas, bis-chalconas e cromonas como possíveis inibidores da proteína de resistência ABCG2. A expressão dessa proteína tanto em células de leucemia quanto em outros tumores torna essas célulasresistentes aos tratamentos convencionais. Neste trabalho a cromona 4a e a bis-chalcona BC20 apresentaram alta atividade inibitória desta proteína comparando-se aos melhores inibidores já estudados e sendo potenciais compostos para ensaios clínicos.Abstract : Cancer is a group of several diseases that have in common the uncontrolled proliferation. Acute lymphoblastic leukemia (ALL) is a malignant disorder caused by the proliferation of lymphoid progenitor cells. Chalcones are essential intermediate compounds in the flavonoids biosynthesis in plants. They have demonstrated a great variety of pharmacological effects including antitumoral. The first part of this work shows the antileukemic activity of three chalcones 2-naphtylacetofenone derivatives. The molecules were named R7, R13 and R15 and their antitumoral activities were evaluated in L1210 cells. The chalcones induced apoptosis in L1210 cells by several mechanisms. Particularly the chalcones R7 and R13 triggered the intrinsic and extrinsic apoptosis pathways as well as the endoplasmic reticulum stress. Conversely, the chalcone R15 triggered only the extrinsic apoptosis and endoplasmic reticulum stress and had no effect on mitochondria. In the second part of this work, the toxicity of these chalcones encapsulated in lipid nanoparticles was evaluated. One of the goals of drug encapsulation is to enhance the pharmacological activity and decrease the side effects. Initially, we evaluated the cytotoxicity of three empty lipid nanoparticles in non-tumoral cells and in vivo, in order to choose a biocompatible system. The three kinds of nanoparticles evaluated were: solid lipid nanoparticles (NLS), composed nanoparticles (NLC) and nanoemulsion (NE). NE was the only system that did not induce toxicity in vitro and was chosen as the vehicle to chalcones incorporation. The encapsulated R7 and R15 induced toxicity in the same level of the free molecules in leukemia cells (L1210) and the encapsulated chalcone R13 induced lower toxicity than free R13, indicating that this molecule probably is not being released from the nanoparticles. After, the antileukemic activity and the toxicity of free and encapsulated chalcones were evaluated in non-tumoral cells (VERO) and in vivo. The encapsulation decreased the toxicity of chalcones in VERO cells and in vivo, mainly by decreasing hepatic inflammation induced by the free molecules in vivo. The results therefore demonstrated that the encapsulation did not improve the antileukemic activity, but reduced their toxicity to non-tumoral cells. The third and last part of this study corresponds to the evaluation of chalcones, bis-chalcones and chromones as potential inhibitors of the resistance protein ABCG2. The high expression of this protein converts these tumors resistant to conventional treatments. As a result of this work the chromone 4a and bis-chalcone BC20 showed high inhibitory activitytoward this protein. This effect is comparable with the best inhibitors that have been studied, resulting in potential compounds for clinical trials

Carbohydrate quality and human health:a series of systematic reviews and meta-analyses

© 2019 Elsevier Ltd Background: Previous systematic reviews and meta-analyses explaining the relationship between carbohydrate quality and health have usually examined a single marker and a limited number of clinical outcomes. We aimed to more precisely quantify the predictive potential of several markers, to determine which markers are most useful, and to establish an evidence base for quantitative recommendations for intakes of dietary fibre. Methods: We did a series of systematic reviews and meta-analyses of prospective studies published from database inception to April 30, 2017, and randomised controlled trials published from database inception to Feb 28, 2018, which reported on indicators of carbohydrate quality and non-communicable disease incidence, mortality, and risk factors. Studies were identified by searches in PubMed, Ovid MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials, and by hand searching of previous publications. We excluded prospective studies and trials reporting on participants with a chronic disease, and weight loss trials or trials involving supplements. Searches, data extraction, and bias assessment were duplicated independently. Robustness of pooled estimates from random-effects models was considered with sensitivity analyses, meta-regression, dose-response testing, and subgroup analyses. The GRADE approach was used to assess quality of evidence. Findings: Just under 135 million person-years of data from 185 prospective studies and 58 clinical trials with 4635 adult participants were included in the analyses. Observational data suggest a 15–30% decrease in all-cause and cardiovascular related mortality, and incidence of coronary heart disease, stroke incidence and mortality, type 2 diabetes, and colorectal cancer when comparing the highest dietary fibre consumers with the lowest consumers Clinical trials show significantly lower bodyweight, systolic blood pressure, and total cholesterol when comparing higher with lower intakes of dietary fibre. Risk reduction associated with a range of critical outcomes was greatest when daily intake of dietary fibre was between 25 g and 29 g. Dose-response curves suggested that higher intakes of dietary fibre could confer even greater benefit to protect against cardiovascular diseases, type 2 diabetes, and colorectal and breast cancer. Similar findings for whole grain intake were observed. Smaller or no risk reductions were found with the observational data when comparing the effects of diets characterised by low rather than higher glycaemic index or load. The certainty of evidence for relationships between carbohydrate quality and critical outcomes was graded as moderate for dietary fibre, low to moderate for whole grains, and low to very low for dietary glycaemic index and glycaemic load. Data relating to other dietary exposures are scarce. Interpretation: Findings from prospective studies and clinical trials associated with relatively high intakes of dietary fibre and whole grains were complementary, and striking dose-response evidence indicates that the relationships to several non-communicable diseases could be causal. Implementation of recommendations to increase dietary fibre intake and to replace refined grains with whole grains is expected to benefit human health. A major strength of the study was the ability to examine key indicators of carbohydrate quality in relation to a range of non-communicable disease outcomes from cohort studies and randomised trials in a single study. Our findings are limited to risk reduction in the population at large rather than those with chronic disease. Funding: Health Research Council of New Zealand, WHO, Riddet Centre of Research Excellence, Healthier Lives National Science Challenge, University of Otago, and the Otago Southland Diabetes Research Trust

Complainant behavioral tone, ambivalent sexism, and perceptions of sexual harassment

Previous research has examined the impact of the law on decisions made about social sexual interactions in the workplace in the context of a variety of individual difference variables including gender of the observer and sexist attitudes, as well as situational factors including legal standard and prior exposure to aggressive and submissive complainants. The current study continued this line of inquiry by testing whether hostile or benevolent sexist attitudes behaved differently under manipulated exposure to aggressive and submissive complainants. Full-time workers watched 1 videotape in which aggressive, submissive, or neutral (i.e., businesslike) women complained that male coworkers sexually harassed them; then, participants viewed a second complainant who always acted in a neutral behavioral tone. In the first case, participants high in hostile sexism who took a reasonable person perspective (but not those with a reasonable woman point of view) and all men who viewed an aggressive complainant found less evidence of harassment. With the second set of allegations, female workers who were exposed to a submissive complainant in the first case found less evidence of harassment against the neutral complainant, suggesting that exposure to a submissive complainant triggered some type of victim blaming in female workers. Policy and training implications are discussed

EFEITO PROTETOR DO ÁCIDO GÁLICO E DODECIL GALATO CONTRA O DANO HEPÁTICO INDUZIDO PELO TETRACLORETO DE CARBONO EM RATOS ALBINOS

O ácido gálico e seus derivados possuem excelente atividade antioxidante, anti-obesidade, anti-asmática e anti-cancerígena. Desde que a hepatotoxicidade induzida por CCl4 é causada principalmente por seus metabólitos reativos, com o presente estudo, objetivou-se avaliar o efeito hepatoprotetor do ácido gálico (GA) e dodecil galato (DGA) em lesões hepáticas induzidas pelo CCl4 em ratos. Foram utilizados 36 ratos Wistar machos divididos em seis grupos. Os ratos do grupo I (controle) receberam apenas veículos (1 mL/kg de DMSO 1% e azeite (3 mL/kg)), já os ratos dos grupos II, III, IV, V e VI foram tratados com CCl4 (30% em óleo de oliva, 3 mL/ kg) por via intraperitoneal duas vezes por semana durante quatro semanas. O GA e o DGA nas doses de 50 e 100 mg/kg foram administrados intragastricamente 30 minutos antes do tratamento com CCl4 aos grupos III, IV, V e VI, respectivamente. O efeito protetor de GA e DGA foi avaliado pela determinação do nível sérico das enzimas hepáticas, bilirrubina total e perfil lipídico, e por meio da investigação das enzimas antioxidantes e do gene p53 (RT-PCR) no tecido hepático. O exame histopatológico do fígado foi também realizado. O GA e o DGA mostraram proteção significativa com redução nos níveis séricos das enzimas ALT e AST e da concentração de bilirrubina total; observou-se também uma redução significativa na peroxidação lipídica hepática e um aumento nos níveis de glutationa. Além disso, verificou-se uma redução nas enzimas antioxidantes hepáticas: catalase, glutationa peroxidase, glutationa-S-transferase e glutationa redutase. Os dados histológicos revelaram uma diminuição na progressão da fibrose hepática nos animais tratados com GA e DGA. Estes resultados indicam que a DGA e GA promovem um aumento da expressão do gene p53, o que induz uma regulação das enzimas antioxidantes, sugerindo um efeito pró-oxidativo do GA e DGA que parece ser seletiva para hepatócitos danificados. Assim, estes resultados sugerem que a GA e DGA tem o potencial para prevenir a fibrose hepática induzida pelotetracloreto de carbono.Palavras-chaves: Enzimas antioxidantes. Hepatotoxicidade. Gene p53

Potencial antitumoral de extratos de Eugenia involucrata DC: uso de produtos regionais para tratar um problema mundial

Artigo apresentado na forma de "banner" na Primeira Mostra Científica e Tecnológica da UFSC CuritibanosNos últimos anos, tem se buscado alternativas mais eficientes e menos tóxicas para a quimioterapia do câncer. Diante disso buscou-se avaliar a atividade antitumoral de extratos da polpa, semente e folha da espécie Eugenia involucrata, nativa do Sul do Brasil. Os extratos vegetais das sementes extraída à 50ºC (S50) e folhas à 22ºC (F22) induziram citotoxicidade em células tumorais PANC-1 demonstrando valores de CC50 iguais a 645 e 596 µg.mL-1, respectivamente, e seletividade para células cancerígenas. A partir da obtenção desses dados, o extrato das folhas passou por um processo de extração líquido-líquido utilizando o solvente hexano (EH), acetato de etila (EAE) e o produto aquoso (EAQ) para selecionar a subfração mais ativa. As frações EAE e EH apresentaram atividade e a fração EAE demonstrou melhor IS, pois não reduziu a viabilidade de células HUVEC até 1000 µg.mL-1. Os resultados obtidos com os extratos foram comparados com o quimioterápico gencitabina, padrão ouro no tratamento do câncer de pâncreas, e se mostraram mais interessantes. As alterações morfológicas características de apoptose foram evidenciadas nas células tratadas com os extratos S50 e F22. O potencial dos extratos em inibir a migração celular e alteração do citoesqueleto também foi avaliado e alguns extratos demonstraram efeito inibitório. Os dados preliminares levam a concluir que a espécie testada apresenta metabólitos de interesse e potencial biológico, o que permite a continuação do estudo em várias frentes de trabalh

mir-181A/B-1 controls thymic selection of treg cells and tunes their suppressive capacity

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1–deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte–associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function

Basic Science Fully Automatic Three-Dimensional Quantitative Analysis of Intracoronary Optical Coherence Tomography: Method and Validation

Objectives and background: Quantitative analysis of intracoronary optical coherence tomography (OCT) image data (QOCT) is currently performed by a time-consuming manual contour tracing process in individual OCT images acquired during a pullback procedure (frame-based method). To get an efficient quantitative analysis process, we developed a fully automatic three-dimensional (3D) lumen contour detection method and evaluated the results against those derived by expert human observers. Methods: The method was developed using Matlab (The Mathworks, Natick, MA). It incorporates a graphical user interface for contour display and, in the selected cases where this might be necessary, editing. OCT image data of 20 randomly selected patients, acquired with a commercially available system (Lightlab imaging, Westford, MA), were pulled from our OCT database for validation. Results: A total of 4,137 OCT images were analyzed. There was no statistically significant difference in mean lumen areas between the two methods (5.03 6 2.16 vs. 5.02 6 2.21 mm 2 ; P 5 0.6, human vs. automated). Regression analysis showed a good correlation with an r value of 0.99. The method requires an average 2-5 sec calculation time per OCT image. In 3% of the detected contours an observer correction was necessary. Conclusion: Fully automatic lumen contour detection in OCT images is feasible with only a select few contours showing an artifact (3%) that can be easily corrected. This QOCT method may be a valuable tool for future coronary imaging studies incorporating OCT

Determinants of participation in a longitudinal two-stage study of the health consequences of the Chornobyl nuclear power plant accident

<p>Abstract</p> <p>Background</p> <p>The determinants of participation in long-term follow-up studies of disasters have rarely been delineated. Even less is known from studies of events that occurred in eastern Europe. We examined the factors associated with participation in a longitudinal two-stage study conducted in Kyiv following the 1986 Chornobyl nuclear power plant accident.</p> <p>Methods</p> <p>Six hundred child-mother dyads (300 evacuees and 300 classmate controls) were initially assessed in 1997 when the children were 11 years old, and followed up in 2005–6 when they were 19 years old. A population control group (304 mothers and 327 children) was added in 2005–6. Each assessment point involved home interviews with the children and mothers (stage 1), followed by medical examinations of the children at a clinic (stage 2). Background characteristics, health status, and Chornobyl risk perceptions were examined.</p> <p>Results</p> <p>The participation rates in the follow-up home interviews were 87.8% for the children (88.6% for evacuees; 87.0% for classmates) and 83.7% for their mothers (86.4% for evacuees and 81.0% for classmates). Children's and mothers' participation was predicted by one another's study participation and attendance at the medical examination at time 1. Mother's participation was also predicted by initial concerns about her child's health, greater psychological distress, and Chornobyl risk perceptions. In 1997, 91.2% of the children had a medical examination (91.7% of evacuees and 90.7% of classmates); in 2005–6, 85.2% were examined (83.0% of evacuees, 87.7% of classmates, 85.0% of population controls). At both times, poor health perceptions were associated with receiving a medical examination. In 2005–6, clinic attendance was also associated with the young adults' risk perceptions, depression or generalized anxiety disorder, lower standard of living, and female gender.</p> <p>Conclusion</p> <p>Despite our low attrition rates, we identified several determinants of selective participation consistent with previous research. Although evacuee status was not associated with participation, Chornobyl risk perceptions were strong predictors of mothers' follow-up participation and attendance at the medical examinations. Understanding selective participation offers valuable insight for future longitudinal disaster studies that integrate psychiatric and medical epidemiologic research.</p

Severe T cell hyporeactivity in ventilated COVID-19 patients correlates with prolonged virus persistence and poor outcomes

Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity.Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity