Avaliação das atividades antitumoral e antioxidante in vitro de extratos de Libidibia ferrea em células de câncer colorretal

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(1)MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. AVALIAÇÃO DAS ATIVIDADES ANTITUMORAL E ANTIOXIDANTE IN VITRO DE EXTRATOS DE Libidibia ferrea EM CÉLULAS DE CÂNCER COLORRETAL. ANDREZA CONCEIÇÃO VÉRAS DE AGUIAR GUERRA. NATAL/RN 2017.

(2) ANDREZA CONCEIÇÃO VÉRAS DE AGUIAR GUERRA. AVALIAÇÃO DAS ATIVIDADES ANTITUMORAL E ANTIOXIDANTE IN VITRO DE EXTRATOS DE Libidibia ferrea EM CÉLULAS DE CÂNCER COLORRETAL. Dissertação apresentada ao Programa de Pós-Graduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito para a obtenção do título de Mestre em Ciências da Saúde.. Orientador: Prof. Dr. Raimundo Fernandes Araújo Junior. NATAL/RN 2017. i.

(3) Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS Guerra, Andreza Conceição Véras de Aguiar. Avaliação das atividades antitumoral e antioxidante in vitro de extratos de Libidibia ferrea em células de câncer colorretal / Andreza Conceição Véras de Aguiar Guerra. - Natal, 2017. 60f.: il. Dissertação (Mestrado) - Programa de Pós-Graduação em Ciências da Saúde. Centro de Ciências da Saúde. Universidade Federal do Rio Grande do Norte. Orientador: Raimundo Fernandes de Araújo Júnior.. 1. Neoplasias Colorretais - Dissertação. 2. Libidibia ferrea Dissertação. 3. Apoptose - Dissertação. 4. Antioxidante Dissertação. I. Araújo Júnior, Raimundo Fernandes de. II. Título. RN/UF/BS-CCS. CDU 616.348-006. ii.

(4) MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. Coordenador do Programa de Pós-Graduação em Ciências da Saúde: Prof. Dr.: Eryvaldo Socrates Tabosa do Egito.. iii.

(5) ANDREZA CONCEIÇÃO VÉRAS DE AGUIAR GUERRA. AVALIAÇÃO DAS ATIVIDADES ANTITUMORAL E ANTIOXIDANTE IN VITRO DE EXTRATOS DE Libidibia ferrea EM CÉLULAS DE CÂNCER COLORRETAL. Aprovada em: 23/06/2017. Banca examinadora:. Presidente da Banca: Prof. Dr. Raimundo Fernandes Araújo Junior (UFRN). Membros da Banca: Prof. Dr. Sergio Adriane Bezerra de Moura Prof. Dr. Jeymesson Raphael Cardoso Vieira. iv.

(6) DEDICATÓRIA. Dedico este trabalhoà minha família, em especial, com todo meu amor e gratidão, aos meus pais Mary e Aldo (in memoriam) e minha avó Fátima por tudo que fizeram por mim e ao meu esposo Maximiliano, pelo apoio e compreensão sempre.. v.

(7) AGRADECIMENTOS. Agradeço primeiramente à Deus por me fortalecer a cada dia nesta caminhada e por me permitir alcançar mais uma conquista. À minha família, que sempre me apoiou, auxiliou e incentivou, sendo minha base e o motivo que me faz ser melhor a cada dia. Ao Prof. Dr. Raimundo Fernandes de Araújo Júnior, expresso meu sincero agradecimento pela confiança, por acreditar em meu potencial e por estar sempre disponívelem me guiar, ajudar e ensinar. À Prof.ª Dr.ª Aurigena Antunes de Araújo, pela valorosa colaboração durante todo o trabalho e pelos ensinamentos que foram essenciais para o desenvolvimento e êxito obtido. Aos alunos que fazem parte da equipe do LAICI, que me acolheram e foram bastante solícitos. Agradeço especialmente às alunas Ana Luiza e Juliana, cuja amizade ultrapassa os limites do laboratório e sempre estiveram dispostas a ajudar, ensinar, a oferecer uma palavra de ânimo, consolo ou mesmo um café, criando momentos descontraídos e deixando mais leve os dias na universidade. Ao Prof. Dr. Hugo Alexandre Rocha, pela colaboração, atenção e por deixar sempre seu laboratório e a sala de cultura de células acessível à nossa equipe. Aos Laboratórios de Imunogenética do Departamento de Bioquímica e de Microscopia do Instituto do Cérebro pelo auxílio e disponibilidade para realização de alguns dos ensaios realizados. Ao Laboratório de Farmacognosia da Universidade Federal de Pernambuco, em especial ao Prof. Dr. Luiz Alberto Soares e à Dr.ª Magda Rhayanny Ferreira pela relevante cooperação durante este trabalho. Ao Programa de Pós-Graduação em Ciências da Saúde, pela oportunidade de realização deste Mestrado. À CAPES pelo apoio financeiro por meio da bolsa de estudos de mestrado que colaborou na conclusão desse projeto. vi.

(8) RESUMO. O câncer colorretal tem se destacado por ser um dos tumores mais freqüentes, com taxas de morbidade e mortalidade expressivos. Na descoberta de novas drogas, produtos derivados de plantas se destacam por ser uma fonte segura e capaz de originar compostos de alta eficiência. Bastante conhecida na medicina popular brasileira, Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea, tem sido utilizada no tratamento de um amplo espectro de condições e na prevenção do câncer. Nesse estudo, extratos etanólicos dos frutos de L. ferrea (a 20T, 40T, 60T e 80T) foram avaliados por 24 h e 48 h pela capacidade de inibição da proliferação celular; indução de apoptose através da avaliação de Bcl-2, caspase-3 e Apaf-1; atividade antioxidante e efeito sobre alvos importantes relacionados a proliferação celular (EGFR e AKT) na linhagem colorretal humana HT-29, por meio de metodologias que envolveram ensaios de citometria de fluxo, espectrofotometria e RT-qPCR. Os resultados demostram que os extratos tiveram atividade antiproliferativa comparado ao controle, indução de apoptose através da via intrínseca e ação de inibição tumoral in vitro com a mediação de alvos importantes na tumorigênese. Além disso, possui efeito antioxidante e anti-peroxidação lipídica, bem como quimioprotetor nas células saudáveis. Portanto, derivados de L. ferrea possuem importantes efeitos anticâncer podendo ser considerados candidatos moleculares promissores para o tratamento do câncer colorretal.. Palavras-chave: Libidibia ferrea. Câncer colorretal. Apoptose. Antioxidante.. vii.

(9) ABSTRACT. Colorectal cancer is noted for being one of the most frequent of tumors, with expressive morbidity and mortality rates. In new drug discovery, plants stand out as a source capable of yielding safe and high-efficiency products. Well known in Brazilian popular medicine, Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz var. ferrea (better known as "ironwood" or "jucá"), has been used to treat a wide spectrum of conditions and to prevent cancer. Using methodologies that involved flow cytometry, spectrophotometry and RT-qPCR assays, ethanolic extracts of the fruits of L. ferrea (20T, 40T, 60T and 80T) were evaluated at 24 h and 48 h for: their ability to inhibit cell proliferation; induce apoptosis through Bcl-2, caspase-3 and Apaf-1; their antioxidant activity and effects on important targets related to cell proliferation (EGFR and AKT) in the HT-29 human colorectal cancer lineage. The results revealed antiproliferative activity as compared to the controls, induction of apoptosis through the intrinsic pathway, and in vitro tumor inhibition activity under the mediation of important targets in tumorigenesis. In addition, L. ferrea revealed antioxidant, lipid peroxidation and chemoprotective effects in healthy cells. Thus, L. ferrea derivatives have important anticancer effects, and may be considered promising candidate for colorectal cancer therapy.. Key words:Libidibia ferrea. Colorectal cancer. Apoptosis. Antioxidant.. viii.

(10) LISTA DE ABREVIATURAS E SIGLAS. 20T (Extrato etanólico a 20% do fruto de Libidibia ferrea) 40T (Extrato etanólico a 40% do fruto de Libidibia ferrea) 60T (Extrato etanólico a 60% do fruto de Libidibia ferrea) 80T (Extrato etanólico a 80% do fruto de Libidibia ferrea) AKT (Proteína Quinase B) Apaf-1 (Fator Apoptótico de Ativação de Protease 1) BCL-2 (Célula B de Linfoma 2) DAPI (4,6-diamidino-2-fenilindol) DMEM (Meio Eagle Modificado de Dulbecco) DTNB (Ácido Dithiobisnitrobenzoico) EDTA (Ácido Etilenodiamino Tetra-acético) EGFR (Receptor do Fator de Crescimento Epidérmico) FITC (Isotiocianato de Fluoresceína) GSH (Glutationa reduzida) HCl (Ácido Clorídrico) HEK-293 (Linhagem de células embrionárias de rim humano) HT-29 (Linhagem de células de câncer colorretal humano) IRI (Índice de Importância Relativa) MDA (Malondialdeído) MTT (Brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio) PBS (Tampão Fosfato Salino) PI (Iodeto de Propídio) RNA (Ácido Ribonucléico) RT-qPCR (Reação de Transcrição Reversa Quantitativa da Cadeia de Polimerase) TCA (Ácido Tricloroacético). ix.

(11) LISTA DE FIGURAS. Figura 1 -Libidibia ferrea e suas estruturas ................................................................................... 14 Figura 2 - Mapa global da carcinogênese ..................................................................................... 15. x.

(12) SUMÁRIO. 1 INTRODUÇÃO ................................................................................................................................ 12 2 JUSTIFICATIVA .............................................................................................................................. 16 3 OBJETIVOS..................................................................................................................................... 17 3.1 Objetivo geral ......................................................................................................................... 17 3.2 Objetivos específicos ........................................................................................................... 17 4 MÉTODO .......................................................................................................................................... 18 4.1 Obtenção dos extratos de Libidibia ferrea e preparo das soluções ........................ 18 4.2 Linhagem celular e cultivo .................................................................................................. 18 4.3 Ensaio de citotoxicidade pelo método do MTT ............................................................. 18 4.4 Avaliação da morte celular por citometria de fluxo ..................................................... 19 4.5 Imunofluorescência .............................................................................................................. 19 4.6 Dosagem de GSH................................................................................................................... 20 4.7 Dosagem de MDA .................................................................................................................. 21 4.8 Real Time RT-qPCR............................................................................................................... 21 4.9 Análise estatística ................................................................................................................. 22 5 ARTIGOS PRODUZIDOS.............................................................................................................. 23 5.1 Full article: Libidibia ferrea presents antiproliferative, apoptotic and antioxidant effects in a colorectal cancer cell line .................................................................................... 25 6 COMENTÁRIOS, CRÍTICAS E CONCLUSÕES ....................................................................... 52 7 REFERÊNCIAS ............................................................................................................................... 54. xi.

(13) 12. 1 INTRODUÇÃO Entende-se que o câncer, condição marcada pela proliferação desordenada de células sutilmente modificadas, é um dos principais problemas de saúde pública enfrentados neste século e apesar dos avanços, os procedimentos terapêuticos disponíveis ainda estão aquém do necessário: são altamente invasivos ou não específicos e muitas vezes estão acompanhados de efeitos secundários e toxicidade para as células1,2. Enquanto isso, os índices de novos casos continuam a aumentar no mundo todo e estima-se que seja superior a 20 milhões por ano até 20253. Dentre os tipos de câncer, o câncer colorretal tem se destacado por ser um dos tumores sólidos mais frequentes, tanto em homens quanto em mulheres, representando 10% da incidência global3. Os fatores etiológicos e mecanismos patogênicos relacionados ao seu desenvolvimento parecem ser complexos e heterogêneos e contribuem para que seja uma das principais causas de morbidade e mortalidade no mundo todo, despertando esforços na investigação de novas estratégias terapêuticas4,5. Atualmente, o desenvolvimento da terapêutica anticâncer tem sido conduzido pela identificação de compostos citotóxicos. Esses agentes têm melhorado as taxas de sobrevivência e a qualidade de vida de pacientes com diferentes tumores, trazendo certas vantagens sobre os convencionais, como menor tempo de administração, mecanismos para superar a resistência aos medicamentos e menor incidência de efeitos adversos6,7. O modelo de triagem convencional é o teste em linhagens celulares, que são uma ferramenta amplamente utilizada devido, entre outros fatores, à sua facilidade de manipulação, caracterização molecular e alto grau de similaridade, sendo excepcionais para o estudo das vias celulares e de genes críticos envolvidos no câncer8. As plantas são consideradas uma das principais fontes de novas entidades químicas biologicamente ativas, com considerável interesse científico e comercial na busca de potenciais fármacos. Para algumas doenças complexas, representam uma fonte extremamente valiosa na produção de drogas inovadoras de alta eficiência 9-11. Estima-se que aproximadamente 40% dos medicamentos disponíveis atualmente foram desenvolvidos, de maneira direta ou indireta a partir de fontes naturais, onde as plantas são as mais utilizadas12..

(14) 13. O Brasil possui a maior diversidade de espécies de plantas no mundo, entre 350.000 a 550.000, porém menos de 10% foram avaliadas no que diz respeito às suas características biológicas13,14. A vegetação da caatinga, em especial, é uma fonte de recursos naturais pouco estudados15. Muitas espécies são amplamente conhecidas, utilizadas empiricamente pela medicina popular, como Libidibia ferrea Martius L. P. Queiroz, também denominada Caesalpinia ferrea e mais conhecida como “pau-ferro” ou “jucá”, uma grande árvore nativa de ampla distribuição no norte e nordeste, pertencente à família Fabaceae (Leguminosae)16. Esta planta é considerada uma das espécies com maior índice de importância relativa (IRI), medida quantitativa baseada no número de propriedades médicas fornecidas por indivíduos de comunidades rurais17. Popularmente, vem sendo usada no tratamento de afecções bronco-pulmonares, distúrbios gastrointestinais, diabetes, doenças renais, inflamações e feridas em geral18-20. Em virtude de seu valor etnomedicinal, o Ministério da Saúde incluiu L. ferrea na lista nacional de plantas medicinais importantes para o sistema de saúde21. Muitos componentes botânicos de L. ferrea são aproveitados como, por exemplo, as folhas, flores, entrecasca e raízes22 (Figura 1). Os frutos, em especial, são vagens achatadas de casca dura e cor marrom escuro utilizados pela população, através de infusões aquosas, na prevenção do câncer23. Na literatura, já foram relatados por possuírem propriedades antimicrobiana24-26, antidiabetes27, além de ser quimiopreventivo23, não apresentar potencial mutagênico28 ou toxicidade reprodutiva29. Apresentam ainda alta atividade antioxidante, descrito pela presença de compostos polifenóis como ácido gálico e epicatequina30. Sobre a composição fitoquímica dos frutos de L. ferrea, além dos compostos fenólicos descritos anteriormente, reporta-se ainda etil galato, metil galato e ácido elágico23,31, bem como a presença de ácidos graxos e terpenoides (ácidos linoleico, palmítico, elaídico, esteárico, além de gama-sisterol e lupenona)32, que são responsáveis por muitas das atividades farmacológicas observadas. Antioxidantes, em especial, produzem uma ação protetora efetiva contra danos oxidativos que estão frequentemente envolvidos na etiologia e progressão de muitas doenças humanas, incluindo o câncer33. Entre os sistemas relacionados à manutenção do equilíbrio redox intracelular, um papel principal é desempenhado pela glutationa (GSH), que também participa de uma multiplicidade de processos, incluindo diferenciação celular, proliferação e apoptose, atraindo a atenção de farmacologistas como um possível alvo de intervenção médica contra o câncer 34..

(15) 14. Figura 1 -Libidibia ferrea e suas estruturas. Adaptado de Melo18.. Um outro parâmetro importante na avaliação da atividade antioxidante é o nível de malondialdeído (MDA), produto final da peroxidação lipídica mediada por radicais livres35. Esse processo é considerado o principal mecanismo de destruição da membrana e lesão celular, tendo sido relatado em vários tipos de câncer, inclusive no câncer colorretal, estando intrinsicamente relacionado ao estresse oxidativo36. Sendo assim, a mensuração da intensidade da peroxidação lipídica é essencial para a melhor compreensão de seus efeitos moleculares deletérios e mutagênicos e na avaliação da resposta dos pacientes à terapia37. A desregulação de vias de sinalização celular relacionadas à apoptose e à proliferação são fundamentais na sobrevivência das células cancerosas e no desenvolvimento tumoral, sendo considerada um dos “hallmakers” ou capacidades biológicas adquiridas38. Há vários fatores na membrana celular que se relacionam com apoptose e crescimento, além do envolvimento de proteínas citoplasmáticas como caspases e AKT (serina/treonina quinase ou proteína quinase B), por exemplo, que por fim atuarão na parada do ciclo celular, inflamação, proliferação, invasão e metástase39(Figura 2). Interferindo-se nos passos de modulação (iniciação,.

(16) 15. promoção, progressão) e nas vias de transdução de sinais associadas, é a maneira mais racional de afetar a carcinogênese40.. Figura 2 - Mapa global da carcinogênese. Adaptado de Bernstein & Lev-Ari31. A apoptose é desencadeada pela ativação cronológica das famílias de caspases iniciadoras (como por ex. caspase 9) e efetoras (como por ex. caspase-3) através de duas vias distintas, mas associadas, conhecidas como via intrínseca e extrínseca. A via intrínseca, em especial, é controlada dominantemente pela família de proteínas Bcl-2 que regulam as decisões relacionadas a sobrevivência celular/morte por agir sob a permeabilização da membrana mitocondrial41, além de ser mediada pela formação inicial do apoptossoma, cujo componente principal é o fator Apaf-142. Outras vias de interesse para estudo do câncer são as vias mediadas por EGFR (receptor do fator de crescimento epidérmico) e AKT que possuem papeis multi-dimensionais no desenvolvimento e crescimento tumoral, estando envolvidas em. numerosas respostas celulares. angiogênse43,44.. inclusive. na. proliferação,. apoptose e.

(17) 16. 2 JUSTIFICATIVA. Apesar do enorme progresso na compreensão da biologia do câncer, os procedimentos terapêuticos ainda são uma exceção e até hoje existem poucos exemplos que levam à cura. Terapias para o câncer colorretal, em especial, são criticamente necessárias e cientificamente desafiadoras. A intervenção cirúrgica é considerada a única modalidade de tratamento com potencial curativo, porém apenas se detectado nos estágios iniciais. Além disso, os pacientes estão sujeitos ao desenvolvimento de metástase, que ocorre em mais de 60% dos casos, e recorrência do tumor. A quimioterapia permanece em grande parte de forma paliativa.45-47 Produtos naturais e, particularmente derivados de plantas, são componentes essenciais na pesquisa e desenvolvimento de medicamentos novos e econômicos. Têm sido reconhecidos durante muitos anos como uma fonte importante de diversidade estrutural, levando a avanços em metodologias sintéticas e promovendo melhorias. nas. propriedades. farmacológica. ou. farmacêutica. de. muitos. agentes9,48,49.Além da ampliação do atendimento médico-farmacêutico na saúde pública, o estudo de plantas medicinais é importante para adquirir conhecimento sobre o potencial farmacológico da diversidade vegetal nativa que permanece subexplorada e auxiliar no desenvolvimento sustentável50. Requisitos como aplicabilidade versátil e segurança terapêutica apresentadas por plantas da caatinga tem atraído a atenção para a busca de novas drogas 51. Espécies popularmente usadas como Libidibia ferrea Martius L. P. Queiroz requerem abordagens etnofarmacológicas que até o momento permanecem escassas. As propriedades anticâncer e antioxidante in vitro, necessitam de uma avaliação mais criteriosa no estudo de possíveis mecanismos de ação dos alvos moleculares para elucidação que conferem tais ações farmacológicas. Diante do exposto, este trabalho permitirá a investigação da potencialidade dessa planta como candidata à fármaco antitumoral e contribuir para inovação na terapia do câncer colorretal..

(18) 17. 3 OBJETIVOS. 3.1 Objetivo geral. Avaliar as atividades antitumoral e antioxidante in vitro induzidas por extratos de Libidibia ferrea frente à linhagem celular de câncer colorretal humano.. 3.2 Objetivos específicos 1) Realizar o screening farmacológico da atividade antiproliferativa in vitro dos extratos etanólicos dos frutos de Libidibia ferrea frente à linhagem de câncer colorretal (HT-29) e à linhagem de células renais embrionárias (HEK-293), através do ensaio de viabilidade celular baseado no sal de tetrazólio MTT. 2) Avaliar o comportamento de morte celular das linhagens HT-29 e HEK-293 através da técnica de citometria de fluxo, pela análise da integridade de membrana, frente aos extratos de L.ferrea. 3) Avaliar a expressão de proteínas mediadoras do processo de morte celular por apoptose (Bcl-2 e caspase-3) nas células HT-29, após tratamento com os extratos de L. ferrea, por microscopia de imunofluorescência. 4) Avaliar a atividade antioxidante das células HT-29 e HEK-293 submetidas ao tratamento pelos extratos de L. ferrea através da dosagem dos níveis de GSH e MDA por espectrofotometria. 5) Avaliar a expressão de genes associados às vias de sinalização AKT, Apaf1 e EGFR nas células HT-29 tratadas com os extratos de L. ferrea através da técnica de Real Time RT-qPCR..

(19) 18. 4 MÉTODO 4.1 Obtenção dos extratos de Libidibia ferrea e preparo das soluções Os extratos etanólicos dos frutos de Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz (Fabaceae) 20T, 40T, 60T e 80T foram obtidos por turbólise na proporção 10% (m/v) utilizando como solvente etanol nas proporções 20, 40, 60 e 80% (v/v), respectivamente. Em seguida, os extratos foram concentrados em rotaevaporador, congelados a -80ºC durante 3 dias e por fim liofilizados. Todo o processo de síntese e análises cromatográficas foi realizado pelo Laboratório de Farmacognosia/Núcleo de Desenvolvimento Analítico e Tecnológico de Fitoterápicos da Universidade Federal de Pernambuco (UFPE) e descrito por Ferreira et al52. As amostras obtidas foram posteriormente pesadas, dissolvidas em meio de cultivo celular para uma concentração inicial de 100 mg/ml e filtradas (filtros estéreis, 0.22 µm) para obtenção das soluções finais para teste.. 4.2 Linhagem celular e cultivo A linhagem de células de câncer colorretal humana HT-29 (HTB-38, ATCC, VA, USA) e a linhagem de células renal embrionária humana HEK-293 (CRL-1573, ATCC, VA, USA) que foi utilizada como controle de parâmetros de citotoxicidade, foram cultivadas em DMEM – Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, MA, EUA) suplementado com 10% de soro fetal bovino e 1% de antibióticos (penicilina/estreptomicina), em uma incubadora à 37˚C, com atmosfera de 5% de CO2. O crescimento celular foi acompanhado com microscópio de luz invertida (NIKON CFI60 - Spectrum Bioengenharia Médica Hospitalar LTDA, BR) e a manutenção das células realizada a cada 3 dias.. 4.3 Ensaio de viabilidade pelo método do MTT A viabilidade e a proliferação das células tratadas com os extratos de Libidibia ferrea foram determinadas através de um ensaio baseado no sal de tretazólio MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio)53. Para tanto, as linhagens celulares HT-29 e HEK-293 foram colocadas em placas de 96 poços, com densidade de 5 x 103 células/poço. Após 24 horas em condições de cultura, foi realizado o.

(20) 19. carenciamento e passado o mesmo período, a aplicação dos extratos etanólicos 20T, 40T, 60T e 80T nas concentrações de 12.5 µg/ml, 25 µg/ml, 50 µg/ml e 100 µg/ml. O tratamento foi avaliado em 24 h e 48 h, através da adição de 100 µl/poço do MTT (1 mg/ml), incubação por 4 h e adição de 100 µl de etanol/poço. As placas foram agitadas e a absorbância obtida em um leitor de microplacas (Epoch - BioTek Instruments Inc, VT, EUA) a 570 nm, com o uso do software Gen5 Data Analysis versão 2.0 (BioTek Instruments Inc, VT, EUA).. 4.4 Avaliação da morte celular por citometria de fluxo. O efeito dos extratos de Libidibia ferrea sobre as células HT-29 e HEK-293 foi determinado por citometria de fluxo com marcação dupla com Anexina V – FITC e Iodeto de Propídio (PI), que permite a identificação de células apoptóticas e necróticas através da perda de integridade de membrana. As linhagens celulares foram dispostas em placas de 6 poços com densidade de 2 x 105 células/poço, com volume total de 2 ml. Após 24 h de incubação em condições de cultura, as células foram tratadas com os extratos 40T, 60T e 80T, pois apresentaram melhor efeito sob a viabilidade das células tumorais. As doses escolhidas para tratamento foram 25 µg/ml e 50 µg/ml, que representaram uma média de inibição da proliferação de 50% (IC50), após a realização de curva dose-resposta. Após os tempos de 24h e 48h, as células foram obtidas através da coleta do sobrenadante dos poços, lavagem com PBS, dissociação enzimática com tripsina e duas etapas de centrifugação a 3000 G a 4ºC. Por fim foram marcadas conforme instruções do kit de Anexina V-FITC/PI (BD Pharmigen, CA, EUA) e analisadas com citômetro BD FACSCanto II (BD Biosciences, CA, EUA) e o software FlowJo, versão 7.6.5 (Tree Star Inc., CA, EUA).. 4.5 Imunofluorescência Para avaliação de alvos de vias de apoptose, as células HT-29 e HEK-293 foram plaqueadas em lamínulas de vidro com densidade celular de 5 x 104 células/poço (para um volume total de 1 ml) e colocadas em placas de 24 poços. Brevemente, passado o tempo de 24 h, as células foram tratadas com os extratos 40T e 60T, dose 25 µg/ml, com base nos que apresentaram melhor ação no ensaio.

(21) 20. anterior. Após cada período de tempo (24h e 48h), foram fixadas com paraformaldeído a 7%, permeabilizadas com Triton X-100/PBS 0.2% e incubadas durante 1 hora em câmera úmida com os anticorpos policlonais de rato anti-Bcl-2 e de coelho anti-caspase-3 (Abcam, CA, EUA), uma lamínula para cada anticorpo, diluídos 1:100 no PBS contendo albumina de soro bovino 5% (Life Technologies, SP, BR). O anticorpo primário foi detectado com o anticorpo secundário Alexa Fluor 488 anti-rato ou anti-coelho (Abcam, CA, EUA) diluídos 1:500 no PBS contendo albumina de soro bovino 5 %, e 4,6-diamidino-2-fenilindol (DAPI) (Life Technologies, SP, BR) diluído 1:200 no PBS contendo albumina de soro bovino 5% foi usado para coloração nuclear. As lamínulas foram examinadas em um microscópio LSM 510 laser scanning (Carl Zeiss, Jena, DE) na objetiva de 40x. As imagens selecionadas foram representativas da maioria das células.. 4.6 Dosagem de GSH. Para avaliar a atividade antioxidante, o nível total de glutationa (GSH) foi determinado através do método de Costa et al54. A obtenção inicial das células foi realizada através do processo descrito por Rahman et. al55. Em resumo, HT-29 foi disposta em placas de 6 poços, com 1 x 106 células/poço, num volume total de 2 ml. No dia seguinte foi realizado o tratamento com os extratos etanólicos 40T e 60T na concentração de 25 µg/ml. Após 24 horas, as células foram removidas, ressuspendidas em PBS e centrifugadas duas vezes por 5 minutos, em 3000 rpm a 4ºC. Em seguida o pellet foi homogeneizado com uma solução de ácido etilenodiamino tetra-acético (EDTA) 0.02M (Sigma-Aldrich, SP, Brazil) para trituração. Após esse processo, 400 µl da suspensão obtida foi então diluída em 80 µl de ácido tricloroacético (TCA) 50% (Vetec, SP, Brazil) e 320 µl de água destilada para centrifugação por 15 minutos em 3000 rpm a 4ºC. Por fim, em cada poço foi aplicado 100 µl do sobrenadante celular, 200 µl do tampão Tris 0.4 M (SigmaAldrich, SP, Brazil) e 20 µl de uma solução de ácido dithiobisnitrobenzoico (DTNB) 0.01 M (Sigma-Aldrich, SP, Brazil), em triplicata, para leitura da absorbância de cada amostra em 412 nm. Os resultados foram expressos em nmol/106 cells..

(22) 21. 4.7 Dosagem de MDA. Para avaliar a peroxidação lipídica, a produção de malondialdeído (MDA) foi medida de acordo com o ensaio descrito por Esterbauer e Cheeseman56. As células passaram pelo mesmo processo inicial de centrifugação descrito no tópico anterior e em seguida foram homogeneizadas com 500 µl de tampão Tris-HCl 20 mM (Trizma HCl, Sigma-Aldrich, SP, Brazil) para trituração. Após esse processo, foram centrifugadas por 20 minutos em 3000 rpm a 4ºC. Então, 375 µl do reagente cromogênico (10.3 mM 1-metil-2-fenilindol em 3:1 acetonitrila) e 112.5 µl de HCl 37% foi adicionado a cada 150 µl de sobrenadante da amostra. Após uma etapa de incubação em banho-maria por 40 min a 45ºC, as amostras foram centrifugadas a 3000 rpm a 4ºC. A absorbância foi medida a 586 nm e os resultados foram expressos em nmol/106 cells.. 4.8 Real Time RT-qPCR O RNA total foi extraído das células HT-29 tratadas por 24 horas com os extratos 40T e 60T (dose 25 µg/ml, em duplicata) com o reagente Trizol (Invitrogen, CA, USA) e SV Total RNA Isolation System (Promega, WI, EUA) segundo as orientações do fabricante. O RNA total sofreu a ação da transcriptase reversa do kit ImProm-IITM Reverse Transcriptase System (Promega, WI, EUA) e a RT-qPCR foi então realizada para a análise quantitativa da expressão de RNA mensageiro (mRNA) com SYBR Green Mix no sistema Applied Biosystems 7500 FAST (Applied Biosystems, CA, EUA), de acordo com o protocolo padrão e os seguintes primers (Integrated DNA Technologies, IA, EUA): AKT (forward: 5’ TCA CCT CTG AGA CCG ACA CC 3’; reverse: 5’ ACT GGC TGA GTA GGA GAA CTG G 3’, temperatura de anelamento: 58.3°C), APAF-1 (forward: 5’ CCT CTC ATT TGC TGA TGT CG 3’; reverse: 5’ TCA CTG CAG ATT TTC ACC AGA 3’, temperatura de anelamento: 56.9°C) e EGF-R (forward: 5’ TGA TAG ACG CAG ATA GTC GCC 3’; reverse: 5’ TCA GGG CAC GGT AGA AGT TG 3’, temperatura de anelamento: 56.9°C). As concentrações finais dos reagentes no mix foram: 5 µl de SYBR Green, 0.7 µl de cada primer, 1.6 µl de água nuclease e 2 µl de cDNA. As condições de PCR padrão foram as seguintes: 50 °C durante 2 min e 95 °C durante 10 min, seguido por quarenta ciclos de 30 s a 95 °C, uma temperatura de anelamento dos primers.

(23) 22. variável durante 30 s e 72 °C durante 1 min. Os valores médios de Cq foram usados para calcular a expressão relativa dos níveis dos genes alvos para os grupos experimentais, em relação aos do grupo controle negativo; os dados de expressão foram normalizados em relação ao gene β-actina humano usando a fórmula 2ΔΔCq57.. 4.9 Análise estatística. Todos os experimentos foram realizados em triplicata. A significância das diferenças entre os grupos foi obtida através da análise de variância (ANOVA) e do teste de Bonferroni (Nível de significância de p<0,05), com o software GraphPad Prism versão 5.0 (GraphPad Software Inc., CA, EUA)..

(24) 23. 5 ARTIGOS PRODUZIDOS. O artigo “Libidibia ferrea presents antiproliferative, apoptotic and antioxidant effects in a colorectal cancer cell line” foi aceito no periódico Biomedicine & Pharmacotherapy que possui fator de impacto 2.326 e Qualis B1 da CAPES para área Medicina II.. Acceptance Letter:.

(25) 24.

(26) ACCEPTED ARTICLE 25. 5.1 Full article: Libidibia ferrea presents antiproliferative, apoptotic and antioxidant effects in a colorectal cancer cell line. Andreza Conceição Véras de Aguiar Guerraa, Luiz Alberto Lira Soaresb, Magda Rhayanny Assunção Ferreirab, Aurigena Antunes de Araújoc, Hugo Alexandre de Oliveira Rochad, Juliana Silva de Medeirose, Rômulo dos Santos Cavalcantea, Raimundo Fernandes de Araújo Júniora,e*. aPost. graduation program in Health Science, Federal University of Rio Grande do. Norte (UFRN), Natal, RN, Cep: 59078-970, Brazil bPost. graduation program in Therapeutic Innovation, Department of Pharmaceutical. Sciences, Federal University of Pernambuco (UFPE), Recife, PE, Cep: 50740-530, Brazil cPost. graduation program in Pharmaceutical Sciences, Department of Biophysics. and Pharmacology, UFRN, Natal, RN, Cep: 59078-970, Brazil dPost. graduation program in Biochemistry, Department of Biochemistry, UFRN,. Natal, RN, Cep: 59078-970, Brazil ePost. graduation program in Functional and Structural Biology, Department of. Morphology, UFRN, Natal, RN, Cep: 59078-970, Brazil. *Corresponding author: Raimundo Fernandes de Araújo Júnior, Department of Morphology, Centre of Biosciences, UFRN, Av. Senador Salgado Filho, S/N, Campus Universitário, Lagoa Nova, 59072-970, Natal, RN, Brazil. E-mail: araujojr@cb.ufrn.br. Abstract Colorectal cancer is noted for being one of the most frequent of tumors, with expressive morbidity and mortality rates. In new drug discovery, plants stand out as a source capable of yielding safe and high-efficiency products. Well known in Brazilian.

(27) 26. popular medicine, Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz var. ferrea (better known as "ironwood" or "jucá"), has been used to treat a wide spectrum of conditions and to prevent cancer. Using methodologies that involved flow cytometry, spectrophotometry and RT-qPCR assays, crude extracts of the fruits of L. ferrea (20T, 40T, 60T and 80T) were evaluated at 24 h and/or 48 h for: their ability to inhibit cell proliferation; induce apoptosis through Bcl-2, caspase-3 and Apaf-1; their antioxidant activity and effects on important targets related to cell proliferation (EGFR and AKT) in the HT-29 human colorectal cancer lineage. The results revealed high antiproliferative potential as compared to the controls, induction of apoptosis through the intrinsic pathway, and probable tumor inhibition activity under the mediation of important targets in tumorigenesis. In addition, L. ferrea revealed antioxidant, lipid peroxidation and chemoprotective effects in healthy cells. Thus, L. ferrea derivatives have important anticancer effects, and may be considered promising candidate for colorectal cancer therapy.. Key words: Libidibia ferrea. Colorectal cancer. Apoptosis. Antioxidant.. 1. Introduction It is understood that cancer, a condition marked by the disorderly proliferation of subtly modified cells, is one of the main public health problems faced in this century. Despite advances, the therapeutic procedures available are still insufficient, they are generally highly invasive or non-specific, and are often accompanied by side effects and cell toxicity [1,2]. Currently, new-case rates continue to rise worldwide and are estimated to be over 20 million per year by 2025 [3]. Among cancers, colorectal cancer has been noted as one of the most frequent in solid tumors, in men and women, representing 10% of the global incidence [3]. The etiological factors and pathogenic mechanisms related to its development are complex and heterogeneous [4,5], this contributes to its being one of the main causes of morbidity and mortality in the world. Natural products, particularly plant derivatives, are essential components in research and development for safe, innovative, economical and high efficiency drugs against complex diseases [6-8]. It is estimated that about 60% of the antitumoral.

(28) 27. drugs available on the market and most of those in the late stages of clinical trials are derived from natural products, mainly from plants [9]. Over the last two decades, public interest and research efforts from scientific and medical communities worldwide has increased expressively, generating a large volume of information including studies on the pharmacological effects, usage, and the development into future medicines of herbs and derivative medicinal phytochemicals as anti-tumor and chemoprevention agents. [10] This leads to growing number of sales of commercialised medicinal herbs and most importantly, growing number of pharmaceutical companies that involve in the research and development of plants as a source for modern medicine. [11] Brazil has one of the largest plant species diversities in the world, yet less than 10% of its species have been evaluated for their biological characteristics [12]. In particular, the vegetation of the caatinga, an extremely threatened biome, as a valuable natural resource has been little studied [13]. Many species however are widely known and used empirically in folk medicine. Thus, we have Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz var. ferrea (Fabaceae) known as Caesalpinia ferrea, and popularly known as "ironwood" or "jucá" [14]. It is used popularly in the treatment of bronchopulmonary disorders, gastrointestinal disorders, diabetes, rheumatism, inflammation, wounds in general, and for prevention of cancers [15]. Most of the botanical components of this plant are harnessed, such as flowers, shells, pods and roots. The fruits have been reported in the literature because they have antimicrobial [16-18], antioxidant [19] and antidiabetic [20] properties, and besides being chemopreventive [21], they show no mutagenic potential [22], or reproductive toxicity [23]. Yet studies exploring their anticancer activity, especially mechanisms of action in cellular signaling pathways are practically nonexistent. Dysregulation of apoptosis and cell proliferation signaling pathways is critical to both the promotion and progression of cancer. Thus, assessment of key points in these pathways is essential for developing new target molecules with effective antineoplastic activity [24,25]. Apoptosis is triggered by chronological activation of the initiator (caspase 9), and effector (caspase-3) families via two distinct but associated pathways known as the intrinsic and extrinsic pathways. The intrinsic pathway in particular is dominated by the Bcl-2 family of proteins that regulate cell survival/death related decisions, acting through permeabilization of the mitochondrial membrane.

(29) 28. [26], in addition to being mediated by initial apoptosome formation, whose main component is the Apaf-1 factor [27]. Other pathways of interest in the study of cancer are the EGFR and AKT (AKT serine/threonine kinase) mediated pathways, which have multidimensional roles in tumor development and growth, and are involved in numerous cellular responses including proliferation, apoptosis and angiogenesis [28,29]. The objectives of this work are to investigate the potential of L. ferrea as an antitumor drug candidate to contribute to colorectal cancer therapy innovation through in vitro evaluations of cell death, antiproliferative effects, and signaling pathways related to intrinsic apoptosis (Bcl-2, caspase-3 and Apaf-1), and to AKT and EGFR expression; and as well, to evaluate L. ferrea’s antioxidant potential. Such bioactive actions presented by L. ferrea may contribute to the availability of new anticancer treatments in the clinic.. 2. Methodology. 2.1 Obtaining Libidibia ferreacrude extracts The crude extracts of fruits from Libidibia ferrea were obtained at 10% (w/v) by turbo extraction (four extractive cycles of 30 sec, interspersed with by 5 min of pause), using ethanol as solvent at 20, 40, 60 and 80% (v/v). The extracts were filtred and concentrated under reduced pressure at 40°C (RV10 Basic, IKA®) to remove the ethanol. The aqueous residues were frozen under -80 °C for three days and then lyophilized (Model L101, Liotop®) to yield the crude extracts. The quantification of chemical markers (ellagic acid and gallic acid) in the crude extracts was carried out by high performance liquid chromatography (HPLC) according to the methodology previously described by Ferreira et al. [30], shown in summary in Table 1..

(30) 29. Table 1 Content of ellagic acid and gallic acid (g%) in crude extracts of fruits from L. ferrea. Sample. Ellagic acid (EA). Gallic acid (GA). 20T. 2.78 ± 1.227 (0.89). 4.43 ± 0.132 (0.24). 40T. 2.89 ± 0.551 (0.39). 3.39 ± 0.268 (0.67). 60T. 2.73 ± 2.213 (1.65). 1.61 ± 0.125 (0.66). 80T. 2.61 ± 0.381 (0.29). 1.84 ± 0.073 (0.34). The results were expressed in g%, as mean ± standard deviation (relative standard deviation).. 2.2 Cell line and cultivation The HT-29 human colorectal cancer cell line and the HEK-293 human embryonic kidney cell line (used as control of cytotoxicity parameters) were obtained from the American Type Culture Collection (Rockville, MD, USA). All cell lines were cultivated in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, MA, USA) supplemented. with. 10%. (v/v). fetal. bovine. serum. and. 1%. antibiotics. (penicillin/streptomycin) in a 37°C incubator with atmosphere of 5% CO2.. 2.3 Viability test Viability and cell proliferation for the Libidibia ferrea extracts were determined by an assay based on the MTT tretazolium salt (3- (4,5-dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide) [31]. To this end, the HT-29 and HEK-293 cell lines were plated in 96-well plates, with a density of 5 x 103 cells/well. After 24 hours under culture conditions, applications of 20T, 40T, 60T and 80T crude extracts at concentrations of 12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL were performed. Treatments were evaluated at 24 h and 48 h by addition of 100 μl/well of MTT (1 mg/mL), incubation for 4 h, and addition of ethanol/well. The plates were shaken and the absorbance obtained in a microplate reader (Epoch - BioTek Instruments Inc, VT, USA) at 570 nm. The Gen5 Data Analysis software version 2.0 (BioTek Instruments Inc, VT, USA) was used.. 2.4 Evaluation of in vitro cell death.

(31) 30. The effect of Libidibia ferrea extracts on HT-29 and HEK-293 cells was determined by double-labeled flow cytometry with Annexin V-FITC and Propidium Iodide (PI), which allows the identification of apoptotic and necrotic cells through loss of membrane integrity. Cell lines were arranged in 6-well plates with a density of 2 x 105 cells/well, at a total volume of 2 mL. After 24 h of incubation under culture conditions, the cells were treated with the 40T, 60T and 80T crude extracts at doses of 25 μg/mL and 50 μg/mL, at 24 h and 48 h. After each period, the cells were obtained by collecting the supernatant from the wells, washing with PBS, trypsinization, and two steps of centrifugation at 3200 rpm at 4°C. Finally, they were labeled according to instructions from the Annexin V-FITC / PI kit (BD Pharmigen, CA, USA), and analyzed with BD FACSCanto II (BD Biosciences, CA, USA) and FlowJo software, version 7.6.5 (Tree Star Inc., CA, USA).. 2.5 Immunofluorescence microscopy For evaluation of apoptosis pathway targets, HT-29 cells was plated on glass coverslips with cell density of 5 x 104 cells/well (for a total volume of 1 mL), and plated in 24-well plates. Briefly, after 24 h the cells were treated with extracts 40T and 60T, being dosed at 25 μg/mL, for 24 h and 48 h. After each period, they were fixed with 7% paraformaldehyde, permeabilized with 0.2% Triton X-100/PBS and incubated for 1 hour in a humid chamber with anti-Bcl-2 and rabbit anti-caspase-3 mouse polyclonal antibodies (Abcam, CA, USA), using one coverslip for each antibody, diluted 1:100 in PBS containing 5% bovine serum albumin (Life Technologies, SP, BR). Primary antibody was detected with Alexa Fluor 488 antimouse or anti-rabbit secondary antibody (Abcam, CA, USA) diluted 1:500 in PBS, containing 5% bovine serum albumin, and 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies, SP, BR), diluted 1:200 in PBS containing 5% bovine serum albumin and used for nuclear staining. The coverslips were examined in an Zeiss Observer Z1 upright microscope for fluorescence (Carl Zeiss, Jena, DE) on the 40x objective. The selected images were representative of most cells.. 2.6 GSH dosage.

(32) 31. To assess antioxidant activity, the total level of glutathione (GSH) was determined using the Costa et al [32] method. Initial cell uptake was performed using the procedure described by Rahman et al [33]. Briefly, HT-29 and HEK-293 cells were plated in 6-well plates, with 1 x 106 cells/well, in 2 mL total volume. On the following day, treatment with extracts 40T and 60T in the concentration of 25 μg/mL was carried out. After 24 hours, the cells were removed, resuspended in PBS and centrifuged twice for 5 minutes at 3000 rpm, and at 4ºC. The pellet was then homogenized with a 0.02 M solution of ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich, SP, Brazil) for grinding. After this process, the suspension obtained was then diluted in 50% trichloroacetic acid (Vetec, SP, Brazil) and distilled water for centrifugation for 15 minutes at 3000 rpm, and at 4°C. Finally, a mix of the cell supernatant,. 0.4. M. Tris buffer. (Sigma-Aldrich,. SP,. Brazil). and. 0.01. M. dithiobisnitrobenzoic acid solution (Sigma-Aldrich, SP, Brazil) were applied to each well, to read the absorbance of each sample at 412 nm. The results were expressed as nmol /106 cells.. 2.7 MDA dosage To evaluate lipid peroxidation, malondialdehyde (MDA production) was measured according to an assay described by Esterbauer and Cheeseman [34]. HT29 cells underwent the same initial centrifugation procedure described above, and were then homogenized with a 20 mM Tris-HCl buffer (Trizma HCl, Sigma-Aldrich, SP, Brazil) for trituration. After this process, they were centrifuged for 20 minutes in 3000 rpm at 4ºC. Afterwards, the chromogenic reagent (10.3 mM 1-methyl-2phenylindole in 3:1 acetonitrile), and a 37% solution of HCl were added to each 150 μL of supernatant sample. After an incubation step in a water bath for 40 min at 45°C, the samples were centrifuged at 3000 rpm at 4°C. The absorbance was measured at 586 nm, and the results were expressed as nmol/106 cells.. 2.8 Real time-qPCR Total RNA was extracted from the HT-29 cells treated for 24 hours with extracts 40T and 60T (25 μg/mL) with the Trizol reagent (Invitrogen, CA, USA) and the SV Total RNA Isolation System (Promega, WI, USA) according to the.

(33) 32. manufacturer's guidelines. First-strand cDNA was synthesized from 1 μg of total RNA with the ImProm-IITM Reverse Transcriptase System (Promega, WI, USA) and Real time-qPCR was then performed for the quantitative analysis of messenger RNA expression (mRNA) with SYBR Green Mix at Applied Biosystems 7500 FAST system (Applied Biosystems, CA, USA), according to the standard protocol, using the following primers (Integrated DNA Technologies, IA, USA): AKT (forward: 5’ TCA CCT CTG AGA CCG ACA CC 3’; reverse: 5’ ACT GGC TGA GTA GGA GAA CTG G 3’, annealing primer temperature: 58.3°C), Apaf-1 (forward: 5’ CCT CTC ATT TGC TGA TGT CG 3’; reverse: 5’ TCA CTG CAG ATT TTC ACC AGA 3’, annealing primer temperature: 56.9°C) e EGF-R (forward: 5’ TGA TAG ACG CAG ATA GTC GCC 3’; reverse: 5’ TCA GGG CAC GGT AGA AGT TG 3’, annealing primer temperature: 56.9°C). The mean Ct values were used to calculate the relative expression of the target gene levels for the cells treated with the extracts, relative to those untreated cells control. The expression data were normalized to the β-actin gene using the formula 2-ΔΔCt [35].. 2.9 Statistical analysis All experiments were performed in triplicate. The significance of the differences between the groups was obtained through analysis of variance (ANOVA), and the Bonferroni test (significance level of p <0.05), with GraphPad Prism software version 7.0 (GraphPad Software Inc., CA, USA).. 3. Results. 3.1 Cell viability To assess the antiproliferative and anticancer potential of the extracts of Libidibia ferrea, HT-29 and HEK-293 cells were treated and evaluated at the 24 hour and 48 hour periods. As observed in Figure 1, during the first 24 hours in HT-29 cell line, inhibition of tumor cell proliferation was observed, with significant action of the 40T, 60T and 80T crude extracts. The 40T extract had levels of cell proliferation inhibition varying between 15% and 25% between doses 25 μg/mL at 100 μg/mL, whereas for the 60T and 80T extracts these levels were higher ranging from 25% to.

(34) 33. about 50% (50% inhibition of proliferation at the dose 25 μg/mL in 60T and 43.7% at the dose of 12.5 μg/mL in 80T). In the same time period, the non-tumor cell line HEK293 showed no cell proliferation inhibition, and the cells exposed to the extracts proliferated in general at rates higher than the untreated cells control (with one exception: a decrease of 15% at 25 μg/mL in 60T). At 48 hours, there was a certain reduction in the proliferation inhibitory effect on the tumor cells, mainly in the 40T and 60T extracts, but the 80T extract still presented proliferation inhibition percentages close to the previous period, ranging from 21.7% to 48.7% between the doses tested. In the non-tumoral lineage, proliferation was observed as either close to or greater than the control. Because the half maximal inhibitory concentration (IC50) on proliferation of the HT-29 cell line was found in the concentration range of 25 μg/mL at 50 μg/mL of the extracts of L. ferrea, those concentrations were selected for the flow cytometry assay..

(35) 34. Fig 1. Effect of Libidibia ferreaextracts on HT-29 and HEK-293 cell line proliferation. (A)24 hour treatment time. (B) 48 hour treatment time. *P < 0.05, **P < 0,01 and ***P<0.001 versus control. CTRL (control, untreated cells), 20T (crude extract 20T), EF40T (crude extract 40T), EF60T (crude extract 60T), EF80T (crude extract 80T).. 3.2 Cell death evaluation For evaluation of cell line deaths after treatment with L. ferrea extracts, an assay with flow cytometry was performed by double labeling with Annexin V - FITC and Propidium Iodide (PI). The selected extracts were the most effective in the cell viability screening (40T, 60T and 80T), and at the intermediate doses (25 μg/mL and 50 μg/mL). In addition, the antineoplastic agent cisplatin (50 μM) was used as the standard drug and tested for comparison purposes. In Figure 2 we observe that during the time period of 24 hours from treatment, all extracts caused cell death by apoptosis in HT-29 tumor cells, and most of the apoptotic cells were found in the initial stage. Among the crude extracts, the 40T at 25 μg/mL presented a higher percentage of cells in apoptosis (38.7%), surpassing even cisplatin, which presented 33.4% of cells in apoptosis. In the non-tumoral lineage HEK-293, there was no statistical difference between the percentages of cells in apoptosis presented by the controls as compared to the extracts. Under the same conditions, the cisplatin antineoplastic presented a significant rate of apoptotic cells (49%). According to Figure 3, at the 48 hour time of treatment in the HT-29 line, half of the extracts tested provoked an increase in the percentage of cells in the apoptosis process in comparison to the previous period, varying between them at 22.4% for 60T at the 25 μg/mL dose, 3.77% for 60T at 50 μg/mL, and 10.79% for 80T at 25 μg/mL). Cisplatin also increased apoptotic cell rates over time (up 28.9%). Of the cells in apoptosis, most were in the early stages. In the non-tumor cell line HEK293, all extracts showed concentrations of viable cells close to or above the control, whereas cisplatin presented viable cells at 43.6%, and an expressive number of cells in apoptosis (54.6%)..

(36) 35. The results are most clearly observed in Figure 4, where they were separated by percentages of cells that were in early apoptosis and late apoptosis in the HT-29 cells and total apoptosis in the HEK-293 cells.. Fig 2. Effect of L. ferrea extracts on HT-29 and HEK-293 cells, after treatment for 24 hours, evaluated by flow cytometry. (A) Control (untreated cells); (B) 40T 25 μg/mL; (C) 40T - 50 μg/mL; (D) 60T - 25 μg/mL; (E) 60T - 50 μg/mL; (F) 80T - 25 μg/mL; (G) 80T - 50 μg/mL; (H) Cisplatin - 50 μM. Dot plots are displayed with Annexin V-FITC (X-axis) e PI (Y-axis). Cells in the upper-left quadrant represent.

(37) 36. nuclear debris (Q1); upper-right quadrant, late apoptotic cells (Q2); lower-right quadrant, early apoptotic cells (Q3); lower-left quadrant, viable cells (Q4).. Fig 3. Effect of L. ferrea extracts on HT-29 and HEK-293 cells, after treatment for 48 hours, evaluated by flow cytometry. (A) Control (untreated cells); (B) 40T 25 μg/mL; (C) 40T - 50 μg/mL; (D) 60T - 25 μg/mL; (E) 60T - 50 μg/mL; (F) 80T - 25 μg/mL; (G) 80T - 50 μg/mL; (H) Cisplatin - 50 μM. Dot plots are displayed with Annexin V-FITC (X-axis) e PI (Y-axis). Cells in the upper-left quadrant represent.

(38) 37. nuclear debris (Q1); upper-right quadrant, late apoptotic cells (Q2); lower-right quadrant, early apoptotic cells (Q3); lower-left quadrant, viable cells (Q4).. Fig 4. Representation of apoptotic cells concentration treated by the extracts of L. ferrea at 24 hour and 48 hour times. Left-side: HT-29 cells; Right-side: HEK-293 cells.. 3.3 Apoptosis activation analyses Activation of important targets of the apoptotic pathway (caspase-3 and Bcl-2) in the HT-29 tumor line, treated with the crude extracts 40T and 60T was performed to study the mechanisms related to the presented apoptosis induction; 25 μg/mL dosage, for 24 h and 48 h (chosen because of better performance in the previous result). The antineoplastic cisplatin was also used for comparison. Representative images are shown in Figure 5 (caspase-3), and Figure 6 (Bcl-2). An intense positive labeling of the effector protease of apoptosis caspase-3 is observed in tumor cells treated with extracts of L. ferrea, as well as with the antineoplastic cisplatin, indicating that the cell death provoked is mediated by an.

(39) 38. apoptotic process. Regarding the anti-apoptotic protein Bcl-2, lower expression was observed for treated cells than for the controls.. Fig 5. Detection of caspase-3 of HT-29 under the effect of L. ferrea extracts in 24 hour and 48 hour times, evaluated by immunofluorescence, with contrast index. (A) Control (untreated cells); (B) Cisplatin - 50 μM; (C) EF40T - 25 μg/mL; (D) EF60T - 25 μg/mL..

(40) 39. Fig 6. Detection of Bcl-2 of HT-29 under the effect of L. ferrea extracts in 24 hour and 48 hour times, evaluated by immunofluorescence, with contrast index. (A) Control (untreated cells); (B) Cisplatin - 50 μM; (C) EF40T - 25 μg/mL; (D) EF60T - 25 μg/mL.. 3.4 GSH and MDA dosages It is highlighted in Figure 7 that glutathione (GSH) levels, an important marker of antioxidant activity, increased by 18% in relation to the control in extract EF60T at 25 μg/mL, which was the sample presenting higher dosages of this protein in the HT29 tumoral lineage. Regarding extracts EF40T and EF60T at the highest dose (50 μg/mL), they presented reduced levels, and we did not find the same effect. Regarding the malondialdehyde oxidative stress marker (MDA), we observed in Figure 8 that all of the extracts at all doses were able to significantly reduce the levels of the protein, thus indicating a protective effect on lipid peroxidation in the HT29 tumor line, being higher than cisplatin..

(41) 40. Fig 7. Effect of L. ferrea extracts on total glutathione (GSH) levels of HT-29 and HEK-293 cells. * P <0.05, ** P <0.01 and *** P <0.001 versus control. CTRL (untreated cells), CIS (cisplatin), 40T (crude extract 40T), 60T (60T crude extract).. Fig 8. Effect of L. ferrea extracts on malondialdehyde (MDA) levels of HT-29 and HEK-293 cells. *P <0.05, **P <0.01 and ***P <0.001 versus control. CTRL (untreated cells), CIS (cisplatin), 40T (crude extract 40T), 60T (crude extract 60T).. 3.5 mRNA expression Based on Figure 9, we see that the AKT gene that is involved in cell survival and proliferation had a slightly reduced expression in the treatment with EF40T at the.

(42) 41. extract dose of 25 μg/mL (4%); extract EF60T with the same dose yielded (26%), a rate still lower than that found with the antineoplastic cisplatin (49%). The Apaf-1 (apoptotic peptidase activating factor 1) gene encoding a cytoplasmic protein that initiates apoptotic events in the intrinsic pathway was found to be reduced in all treatments. Epidermal growth factor receptor (EGFR), involved in the pathogenesis and progression of different carcinomas, showed a reduction in expression as compared to the control: 29% for the EF40T extract, 48% for the EF60T extract and 53% for cisplatin.. Fig 9.Effect of L. ferrea extracts on the expression of AKT, Apaf-1 and EGFR mRNAs in HT-29. *P < 0.05, **P < 0,01 e ***P<0.001 versus control. CTRL (untreated cells), CIS (cisplatin), EF40T (40T crude extract), EF60T (60T crude extract).. 4. Discussion In addition to expanding medical-pharmacological care in public health, the study of medicinal plants is important for acquiring knowledge about the pharmacological potential of a native plant diversity that remains under exploited, and helps to ensure its rational exploitation [13]. The versatility, applicability and therapeutic safety presented by caatinga plants have attracted great attention to them in the search for new drugs [36]. Popular species such as Libidibia ferrea.

(43) 42. Martius L. P. Queiroz require ethno-pharmacological approaches, and these have so far remained scarce, especially regarding anticancer activity. Crude and fractionated extracts obtained from the fruits of L. ferrea tested by Freitas et al [37] on human cancer cell lines NCI-H292 (mucoepidermoid carcinoma of the lung), HEP-2 (squamous cell carcinoma of the larynx), and solid tumor sarcoma 180 presented no significant antitumor activity or inhibition of cell proliferation. In the present study, for the HT-29 tumoral lineage (human colorectal adenocarcinoma), proliferation inhibition by the 40T, 60T and 80T crude extracts was significant in the first observed hours. In addition, during the same time period, the same extracts did not generally present toxicity in the non-tumor cell line HEK-293 (human embryonic renal cell). Alterations to apoptosis death process related pathways and consequent cell resistance to this mechanism are the main factors responsible for the onset of tumorigenesis, considered one of the fundamental hallmarks of cancer [38]. Despite causing the problem, apoptosis plays an important role in cancer treatment strategies through the eradication of transformed cells, and resistance to conventional treatments [39]. Induction of apoptosis by L. ferrea has been described previously by Nozaki et al [40], using acetone stem extract in human acute myeloid leukemia (HL-60) lineage. In this work, all extracts from the L. ferrea fruit tested in the HT-29 line induced cell death by apoptosis, corroborating their results. The apoptotic activity exhibited in the present study has a profile suggestive of affecting the intrinsic apoptosis pathway, a common target of most anticancer agents [41], through activation of caspase-3 and reduction of Bcl-2 levels. This pathway results from increased mitochondrial membrane permeability (with inhibition of antiapoptotic regulator Bcl-2), and the release of pro-apoptogenic factors in the cytoplasm, promoting the activation of effector enzymes (such as caspase-3), which act on a broad spectrum of substrates, resulting in cell death [42]. Most of the apoptotic cells in this work were detected in the initial stage, characterized by phosphatidylserine exposure (an event that precedes cell membrane permeabilization), cellular shrinkage, and nuclear condensation. In addition, the collapse of the mitochondrial transmembrane potential has also been reported as an early event in the apoptotic process, being present in many cell types.

(44) 43. in apoptosis that culminate in the release of several apoptotic proteins, independently of the stimulus [43]. Therapies for colorectal cancer designed to stimulate apoptosis, as presented here, play a critical role in controlling its development and progression, as well as improving response to chemotherapy and radiation [44]. To assess antioxidant status, dosages were performed for analysis of GSH (glutathione) and MDA (malondialdehyde) levels. We observed that the EF60T extract, at the lowest dose tested presented a significant increase in the level of GSH, an intracellular peptide that exerts, among other functions, detoxification, elimination of free radicals, maintenance of the thiol state, and cellular proliferation modulation [45]. As for the MDA lipid peroxidation marker, all of the extracts at all doses presented reductions, which is an important sign for prevention of MDA’s mutagenic and genotoxic activities in tumor cells [46]. All in all, the extract stands out for presenting potential antioxidant activity. The antioxidant activities of L. ferrea fruits were first described by Silva et al [47] through several in vitro assays. Later, Barros et al [19] also described augmented antioxidant activity in addition to hepatoprotection in mice. The expression of certain relevant genes to tumor signaling machinery was evaluated in this work through identification of mRNA-targets (EGFR, AKT and APAF-1). We found that levels of the EGFR receptor (involved in growth, invasion and metastasis of epithelial tumors and particularly important in the development of colorectal tumors) [48] were reduced after treatment with the extracts, indicating that L. ferrea interferes with this pathway and is effective in tumor inhibition. Expression of AKT was varied, with reduced expression for EF40T and increased expression for EF60T. Interestingly, some studies have shown that AKT is not a single-function kinase and may facilitate, rather than inhibit, cell death under certain conditions [49], thus being involved both in inducing death and cell survival. The same scenario of opposite functions in the same protein may also involve Apaf-1 factor, whose expression was reduced in cells treated with L. ferrea. Although this protein is known for its apoptotic role in the intrinsic pathway, recent work has suggested additional non-apoptotic functions including a pro-survival role that needs to be better investigated [50]. Through various mechanisms, cancer itself, through induction of pro-survival signals such as the AKT activation pathway bypasses activation of the intrinsic.

(45) 44. pathway as in blockade of apoptosome formation and consequently of Apaf-1. Thus, strategies to bypass resistance, through therapeutic restoration of apoptotic pathways as conducted by interactions with the tumor microenvironment, provide new promise for cancer patients in clinical treatment [51]. Therefore, the differing expression profiles presented in this study from the extracts of L. ferrea may be related to different activities in the mediation of important pathways in tumor development, that culminate in cell death stimuli, as already verified in the previous results. Mediation of the AKT pathway, in addition to influencing apoptosis induction impacts oxidization capacity. Ramos et al [52] showed that apoptosis caused by treatment with low concentrations of arsenic trioxide in human myelogenous leukemia cells (HL-60) was potentiated by the use of AKT pathway inhibitors, which also led to the reduction of levels of GSH causing intracellular oxidation. The results demonstrated that AKT signaling pathway activity is necessary to maintain normal GSH metabolism in cells. We observed in the current study that there is also synchronization between AKT expression levels and intracellular GSH levels: increased AKT expression is accompanied by increased GSH and vice versa. The main constituents found in the L. ferrea extracts tested were the hydrolyzable tannins: gallic acid (GA) and ellagic acid (EA) (30). GA is a phenolic compound naturally present in a wide variety of fruits and plants exhibiting various pharmacological activities such as antimicrobial, antitumor, anti-inflammatory, antioxidant, antidepressant, antidiabetic, anxiolytic, and others [53]. The acid is associated to inhibition in various cancer cell lines including human colorectal carcinoma cells through apoptosis induction by different mechanisms such as regulation of apoptotic and anti-apoptotic proteins [54]. It is also well known for its efficient protection against oxidative damage, maintenance of endogenous defense, and chiefly, prevention of lipid peroxidation [55]. EA is a dimeric derivative of gallic acid present in the plant vacuole and, like other polyphenols; it has a wide range of biological activities such as antioxidant, anti-inflammatory, prebiotic and anticancer functions, and the latter by selectively inhibiting growth in differring tumor types, including inducing apoptosis in human colon adenocarcinoma cells via the mitochondrial pathway [56]..

(46) 45. Among the results reported, it is important to highlight the protective effect of L. ferrea extracts on non-tumor HEK-293 cells, which presented high rates of cell proliferation and viable cells. In vitro evaluations by Nakamura et al [57] have demonstrated the chemopreventive effects of L. ferrea through the presence of ellagic acid in its composition: three adjacent hydroxyl phenolic groups were responsible for cytotoxicity, and the carboxyl group appeared to be implicated in the distinction between normal and tumor cells. These results are consistent with studies by Araújo Júnior et al [58,59], in the sense that many plant derivatives may act both in inhibiting the proliferation and viability of tumor cells, and in mediating a protective effect on healthy cells.. 5. Conclusions The present results demonstrate that L. ferrea has antiproliferative activity in HT-29 cells with induction of apoptosis in association with mitochondrial effects in the intrinsic pathway (via caspase-3 activation, negative regulation of Bcl-2 and Reduction of Apaf-1), this as well as acting in the expression of important targets in the process of colorectal cancer development such as EGFR and AKT, with probable tumor inhibition. L. ferrea also has antioxidant and lipid peroxidation inhibiting effects, together with chemopreventive action in healthy renal cells. Taken as such, L. ferrea is a promising candidate for cancer therapy with both efficacy and selectivity as potential characteristics.. Acknowledgments The authors thank the Pharmacognosy Laboratory of Pernambuco Federal University (UFPE); the cell culture room of the Department of Biochemistry, the Health Sciences Graduate Program, and the Brain Institute Microscopy Laboratory at Rio Grande do Norte Federal University (UFRN).. References [1] V. Pavet, M.M. Portal, J.C. Moulin, R. Herbrecht, H. Gronemeyer, Towards novel paradigms for cancer therapy, Oncogene 30 (2011) 01-20..

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