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Keywords: [19-NαC]-crourorb A1, cytotoxicity, selectivity, apoptosis and cell cycle

À Coordenação

Croton urucurana Baillon (Euphorbiaceae), est une plante très connue et utilisée en médecine traditionnelle au Brésil, la «sangra-d'água» retrouvée dans l'État du εato Grosso do Sul (région Centre-Ouest du Brésil). Son latex est abondamment utilisé en médecine traditionnelle dans le traitement de plusieurs types de cancer. En dépit de son importance ethnopharmacologique, on ne trouve aucune étude sur sa composition chimique ou sur sa potentielle activité anti-cancéreuse. δ'objectif de ce travail a été d'évaluer l'activité cytotoxique des extraits totaux, fractions d'extraits et de molécules purifiées, isolées du latex de C. urucurana. Nous avons étudié plus particulièrement les effets du crourorb A1 sur la viabilité, l'apoptose, le cycle cellulaire et la migration de cellules issues de carcinome hépatocellulaire humaine . Nous avons également évalué l'effet du crourorb A1sur la viabilité des cellules Huh-7 cultivées dans les gels de collagène en 3D. L' activité cytotoxique ont été réalisées par le test à la sulforhodamine B sur six lignées cellulaire cancéreuses et sur une lignée cellulaire non cancéreuse de fibroblastes murins, les cellules NIH/3T3. L'étude phytochimique de la fraction 'acétate d'éthyle' du latex de C. urucurana bioguidé a permis d'obtenir un nouveau cyclopeptide que nous avons nommé [19-NαC]-crourorb A1. Le crourorb présente une cytotoxicité puissante sur la lignée cellulaire cancéreuse NCI-ADR/RES (ovaire, le phénotype de multirésistance) à un niveau équivalent à celle de la doxorubicine (témoin positif). Ce composé est de plus pratiquement inactif (GI 50 = >250 µg/mL) sur la lignée non cancéreuse NIH/3T3. L'évaluation de la cytotoxicité in vitro du crourorb A1 (test wst1) sur les cellules Huh-7 a démontré que la viabilité des cellules est inhibée de manière dose et temps-dépendant. Nous avons mesuré une forte activation des caspases 3 et 7 et une nette augmentation de l'expression des protéines pro-apoptotiques (Bak, Bid, Bax, Puma, Bim et Bad) ainsi que de la caspase 3 clivée par le crourorb A1. Nous avons également déterminé que la voie de signalisation JNK-MAPkinase semble nécessaire à l'induction de la mort cellulaire des cellules Huh-7 traitées au crourorb A1. Le crourob A1 induit un arrêt du cycle cellulaire en phase G 2 /M. En parallèle, les expressions de la cycline-dépendante kinase CDK1, des cyclines B1 et D1 sont augmentées après traitement au crourorb A1. De plus, le crourorb A1 a un effet inhibiteur significatif de la migration des cellules Huh-7, en présence ou absence de mitomycine C. Nous avons également confirmé, l'effet inhibiteur du crourob A1 sur la viabilité des cellules Huh7 cultivées en 3D dans les gels de collagène 1 et démontré que les cellules cultivées en 3D semblent plus résistantes au crourorb A1 par rapport aux cultures en monocouche 2D. 

INTRODUÇÃO

Câncer é a designação dada a um grupo de doenças caracterizadas pelo acúmulo de alterações genéticas e epigenéticas que resulta na perda do controle da proliferação celular e na capacidade dessas células invadirem tecidos próximos ou migrarem para outros locais do organismo, caracterizando as metástases [START_REF] Hanahan | Hallmarks of Cancer: the Next Generation[END_REF][START_REF] Hanahan | Hallmarks of Cancer: the Next Generation[END_REF]PINTO;FELZENSWALB, 2003).

O câncer é apontado como um dos principais problemas de saúde pública no mundo (WORLD HEALTH ORGANIZATION, 2015). Em decorrência da magnitude da doença, uma parte significativa das pesquisas de produtos naturais, especialmente de plantas medicinais tem-se centrado na busca de substâncias para prevenir ou tratar o câncer [START_REF] Montenegro | Contribuição dos Produtos Naturais como Fonte de Novos Fármacos Anticâncer: Estudos no Laboratório Nacional de Oncologia Experimental da Universidade Federal do Ceará[END_REF]CRAGG;NEWMAN, 2013;[START_REF] Houghton | The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity[END_REF].

As plantas medicinais têm sido sempre uma fonte promissora de substâncias antitumorais em função da diversidade de espécies vegetais disponíveis para investigação fitoquímica e por proporcionar grandes chances de obtenção de uma molécula protótipo devido à riqueza de constituintes presentes nestas espécies (YUNES; [START_REF] Yunes | Plantas medicinais sob a ótica da química medicinal moderna[END_REF]. Muitas substâncias em amplo uso clínico utilizadas no tratamento do câncer foram descobertas por meio de investigação de extratos de plantas em programas de triagem de novos compostos (CRAGG; [START_REF] Cragg | Plants as a source of anti-cancer agents[END_REF].

Nesse contexto, a medicina popular constitui uma valiosa fonte de informações que servem como ponto de partida para seleção de plantas já empregadas para fins medicinais, na busca de identificar e isolar substâncias com atividades biológicas. Os alcaloides da vinca, vincristina e vimblastina, exercem suas propriedades anticâncer inibindo a mitose através da ligação com a tubulina, interrompendo, deste modo, a divisão celular na metáfase [START_REF] Heinrich | Fundamentals of Pharmacognosy and Phytotherapy[END_REF]. As epipodofilotoxinas (etoposideo e teniposideo) se ligam a tubulina, causando quebras na cadeia de DNA durante a fase G2 do ciclo celular por inibição irreversível da topoisomerase II [START_REF] Gordaliza | Natural products as leads to anticancer drugs[END_REF]. O paclitaxel mais comumente conhecido como taxol, estabiliza os microtúbulos e conduz à parada mitótica [START_REF] Gurib-Fakim | Medicinal plants: Traditions of yesterday and drugs of tomorrow[END_REF][START_REF] Wani | Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia[END_REF]. Outra adição importante ao arsenal de drogas anticâncer é a classe de agentes clinicamente ativos derivados da camptotecina, o irinotecano e topotecano, que atuam inibindo a enzima topoisomerase I resultando no rompimento da dupla fita de DNA e consequente interrupção da replicação e transcrição [START_REF] Granada | Emprego de sistemas de liberação como estratégia para melhorar as propriedades terapêuticas de fármacos de origem natural: o exemplo da camptotecina e seus derivados[END_REF][START_REF] Mann | Natural products in cancer chemotherapy: past, present and future[END_REF][START_REF] Rahier | Camptothecin and its analogs[END_REF][START_REF] Rahier | Camptothecin and its analogs[END_REF][START_REF] Rahier | Camptothecin and its analogs[END_REF].

Entretanto, grande parte das drogas anticâncer disponíveis apresentam diversas limitações terapêuticas, tais como a toxicidade sistêmica, baixa especificidade para células tumorais, resistência do organismo à droga e rápido metabolismo, além de vários efeitos colaterais (JOHNSTONE; RUEFLI; [START_REF] Johnstone | Apoptosis: a link between cancer genetics and chemotherapy[END_REF]. Nesse sentido, a busca de novos protótipos naturais com atividade anticâncer tem sido cada vez mais estimulada, visando à descoberta de moléculas que atuem com mecanismos específicos para cada tipo de câncer e apresentem seletividade às células tumorais, com efeitos colaterais mínimos sobre o tecido normal; ou ainda, moléculas que impeçam o avanço da doença [START_REF] Brandão | Química e farmacologia de quimioterápicos antineoplásicos derivados de plantas[END_REF].

Os ciclopeptídeos derivados de plantas têm emergido como uma nova classe de biomoléculas com potencial atividade anticâncer (FANG et al., 2013;MISHRA et al., 2014;TAN;ZHOU, 2006, YUE et al., 2011). Estudos têm demonstrado que estas moléculas inibem o crescimento de células tumorais através da parada do ciclo celular e indução de apoptose [START_REF] Ma | Peptides from Plants: A New Source for Antitumor Drug Research[END_REF]. MISHRA et al., 2014;. YUE et al., 2011) [START_REF] Figueiredo | A new cytotoxic -carboline alkaloid from Galianthe thalictroides[END_REF][START_REF] Figueiredo | Further constituents of Galianthe thalictroides (Rubiaceae) and inhibition of DNA topoisomerases I and IIα by its cytotoxiccarboline alkaloids[END_REF][START_REF] Garcez | Cytotoxic and genotoxic butanolides and lignans from Aiouea trinervis[END_REF][START_REF] Garcez | Bioactive flavonoids and triterpenes from Terminalia fagifolia (Combretaceae)[END_REF][START_REF] Garcez | Cytotoxic aporphine alkaloids from Ocotea acutifolia[END_REF][START_REF] Matos | Antineoplastic activity of selected constituents of Duguetia glabriuscula[END_REF][START_REF] Silva | Isolamento e avaliaçao da atividade citotóxica de alguns alcalóides oxaporfínicos obtidos de Annonaceae[END_REF][START_REF] Silva | Chemical composition and cytotoxic activity of the root essential oil from Jatropha ribifolia (Pohl) Baill (Euphorbiaceae)[END_REF][START_REF] Simionatto | Bioactivity and chemical Ccomposition of the essential oils of Croton urucurana Baillon (Euphorbiaceae)[END_REF][START_REF] Simionatto | Chemical compositon and cytotoxic activiy of leaves essencial oil from Mangifera indica var. coquinho.(Anacardiaceae)[END_REF][START_REF] Simionatto | Chemical composition and biological activities of leaves essential oil from Schinus molle (Anacardiaceae)[END_REF].

Plantas medicinais e o gênero Croton

O gênero Croton é um dos maiores da família Euphorbiaceae, com cerca de 1300 espécies de árvores, arbustos e ervas distribuídas em regiões tropicais e subtropicais do mundo (WEBSTER, 1993;SALATINO;SALATINO;NEGRI, 2007). O gênero apresenta grande diversidade morfológica e ocupa uma grande variedade de habitats [START_REF] Motta | Antiproliferative activity and constituents of leaf extracts of Crotonsphaerogynus Baill. (Euphorbiaceae)[END_REF]; sendo classificado como o 11º maior gênero de angiospermas [START_REF] Wiley | G. History and concepts of big plant genera[END_REF]. Na América do Sul, o Brasil destaca-se por possuir a maior concentração de espécies do gênero (356 espécies) (LIMA; PIRANI, 2008).

Assim como na maioria das espécies de Euphorbiaceae, Croton pode conter látex, que é de cor vermelha em algumas espécies, uma característica normalmente associada a propriedades medicamentosas (RISCO et al., 2003;SANDOVAL et al., 2002). Espécies do gênero Croton são amplamente utilizadas na medicina popular. , 1985, apud LIMA et al., 2008;;[START_REF] Lorenzi | Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas nativas do Brasil[END_REF].

Caracteriza-se como uma árvore de 6-8 m de altura, de copa aberta e tronco claro com cerca de 20 cm de diâmetro. Apresenta folhas em formato de coração que adquirem coloração vermelho-amarelada quando estão para cair, principalmente no outono, e flores pequenas e esbranquiçadas, dispostas em inflorescências espigadas terminais (LORENZI; MATOS, 2008) (Figura 3). 2008). Em espanhol são chamadas de "sangre de drago" ou "sangre de grado", traduzido para inglês como "dragon's blood" e "blood of the tree" (SALATINO; SALATINO; NEGRI, 2007) (Figura 4).

Figura 4 -Extração do látex de "sangra d'água". Tronco com corte "espinha de peixe".

Foto: Cândido-Bacani, 2014.

A espécie Croton urucurana é usada na medicina popular devido a seus efeitos analgésico e anti-inflamatório. É usada também para tratar infecções, dor e para acelerar o processo de cicatrização de feridas e no tratamento de reumatismo, câncer e outras doenças (SALATINO; SALATINO; NEGRI, 2007). Popularmente duas partes da planta são utilizadas, as cascas do caule e o látex (SALATINO; SALATINO;NEGRI, 2007;[START_REF] Simionatto | Chemical Composition and eEvaluation of antibacterial and antioxidant activities of the essential oil of Croton urucurana Baillon (Euphorbiaceae) stem bark[END_REF]. Esta espécie possui interessantes atividades farmacológicas descritas, incluindo cicatrizante [START_REF] Lorenzi | Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas nativas do Brasil[END_REF][START_REF] Silva | Estudo Farmacognóstico de Croton urucurana Baillon (sangra d'água)[END_REF]), antidiarreica (GURGEL et al., 2001), antifúngica (GURGEL et al., 2005), anti-hemorrágica (ESMERALDINO; SOUZA; SAMPAIO, 2005), antioxidante [START_REF] Simionatto | Chemical Composition and eEvaluation of antibacterial and antioxidant activities of the essential oil of Croton urucurana Baillon (Euphorbiaceae) stem bark[END_REF], antinoceptiva [START_REF] Rao | Dragon's blood from Croton urucurana (Baill.) attenuates visceral nociception in mice[END_REF], antimicrobiana (PERES et al., 1997;[START_REF] Simionatto | Chemical Composition and eEvaluation of antibacterial and antioxidant activities of the essential oil of Croton urucurana Baillon (Euphorbiaceae) stem bark[END_REF]OLIVEIRA et al., 2008), citotóxica (SIMIONATTO et al., 2009), inseticida (SILVA et al., 2009), antiinflamatória (MATTOS, 2001) e antiulcerogênica (CORDEIRO et al., 2012;[START_REF] Silva | Estudo Farmacognóstico de Croton urucurana Baillon (sangra d'água)[END_REF]. Do ponto de vista da ecologia da restauração, esta espécie desempenha um papel muito importante na recuperação de áreas ciliares degradadas (LIMA et al., 2008;[START_REF] Lorenzi | Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas nativas do Brasil[END_REF].

Estudos toxicológicos mostraram que o látex de C. urucurana apresenta alta toxicidade subcrônica por via oral em ratos Wistar [START_REF] Silva | Estudo Farmacognóstico de Croton urucurana Baillon (sangra d'água)[END_REF]. [START_REF] Silva | Estudo Farmacognóstico de Croton urucurana Baillon (sangra d'água)[END_REF] demostrou que animais tratados com látex na dose de (PERES et al., 1997;PERES et al., 1998a;PERES et al., 1998b). É referido também o isolamento do ácido acetilaleuritólico e catequinas ativos contra Staphylococcus aureus e Salmonella typhimurium (PERES et al., 1997).

A análise dos constituintes químicos do látex revelou a presença de saponinas, esteroides, alcaloides, taninos pirogálicos, taninos catequinicos, flavonoides e flavononas antocianinas e antocianidinas [START_REF] Gurgel | Avaliação experimental da atividade antidiarréica do látex do Croton urucurana Baill. 2000. 146f. Dissertação[END_REF]. [START_REF] Milo | Characterization of a fucoarabinogalactan, the main polysaccharide from the gum exudate of Croton urucurana[END_REF] 

Cultura de células

O cultivo celular é caracterizado por permitir a manutenção de células vivas in vitro a partir de explantes primários ou de suspensão de células dispersas, que podem ser cultivadas em monocamadas aderentes em um substrato sólido, ou em suspensão em meio de cultura de células, quando as células não aderem ao substrato (FRESHNEY, 2010). As culturas em suspensão são derivadas de tecidos que não necessitam de ancoragem para proliferar e sobreviver, tais como às células hematopoiéticas, às linhagens transformadas ou às células de tecido tumoral. As células aderentes são oriundas de tecidos duros e, por isso, são dependentes de ancoragem e requerem uma superfície de contato para crescimento e proliferação (ALVEZ; GUIMARÃES, 2010).

A técnica de cultura de células foi estabelecida pela primeira vez no início do século XX com Harrison, em 1907, e Carrel, em 1912. Essa técnica foi desenvolvida como um método para estudar o comportamento de células animais livres de restrições impostas pelo ambiente que podem surgir, in vivo, tanto durante a

homeostase normal e sob a tensão de uma experiência laboratorial (FRESHNEY, 2010). Nos anos de 1940 foi estabelecida a primeira linhagem de células normais de roedores e, em 1951, a partir dos trabalhos desenvolvidos por George Gey, a primeira linhagem de células de câncer humano contínuo (HeLa). A linhagem HeLa foi estabelecida a partir de células obtidas de câncer de colo uterino de uma mulher de mais de 50 anos [START_REF] Halama | Metabolomics in cell culture-A strategy to study crucial metabolic pathways in cancer development and the response to treatment[END_REF][START_REF] Masters | HeLa cells 50 years on: the good, the bad and the ugly[END_REF].

Com o aprimoramento da cultura de células foi possível, em meados da década de 80, a manutenção de linhagens celulares oriundas de tumores humanos de diferentes órgãos (SHOEMAKER, 2006). Essas linhagens celulares são utilizadas em modelos laboratoriais e são fundamentais para estudar a biologia do câncer e para testar a terapêutica e a eficácia de compostos com atividade anticâncer (SHARMA; HABER; SETTLEMAN, 2010). Tais linhagens celulares podem ser facilmente encontradas em diversos bancos de cultura de células (Figura 5). Por exemplo, a American Type Culture Collection (ATCC), criada em 1962, é um dos maiores centros de recursos biológicos que oferecem linhagens celulares de mais de 150 espécies diferentes, incluindo cerca de 4000 linhagens de células humanas (HALAMA, 2014).

Figura 5 -Bancos fornecedores de linhagens celulares. O método convencional de cultura de células em monocamadas, chamado de cultura em duas dimensões (2D) tem, segundo Rebello ( 2014) "contribuído enormemente para o conhecimento de diversos aspectos da fisiologia celular, da ação de fármacos e de hormônios e da interação com microrganismos". O baixo custo e a rapidez na realização de diferentes testes em um reduzido intervalo de tempo fizeram da cultura de células em 2D um componente essencial dos programas de descoberta de drogas, especialmente no screening de compostos com potencial atividade anticâncer [START_REF] Giese | Unraveling novel intracellular pathways in cell-based assays[END_REF]MONKS et al., 1991). As principais vantagens do uso de cultura de células incluem, o controle físico-químico do ambiente (pH, temperatura, pressão osmótica, tensão de CO 2 e O 2 ) e a manutenção constante das condições fisiológicas, além de reduzir o número de ensaios com animais (FRESHNEY, 2010).

No entanto, as limitações da cultura de células em 2D tornam-se cada vez mais reconhecidas [START_REF] Sun | Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents[END_REF]. Nesse modelo de cultura, a adesão célulacélula e célula-matriz são reduzidas devido às restrições geométricas e mecânicas altamente antinaturais impostas sobre as células que não possuem as características (heterogeneidade e arquitetura tridimensional) de um tecido in vivo, uma vez que seu meio nutricional e hormonal está modificado (ALVEZ; [START_REF] Karp | Biologie cellulaire et moléculaire[END_REF][START_REF] Sun | Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents[END_REF]. Por conseguinte, as condições da cultura podem não refletir a situação in vivo para citotoxicidade e/ou resistência a drogas [START_REF] Sun | Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents[END_REF].

GUIMARÃES

Para contornar essas limitações, modelos de cultura de células cultivadas em ambiente tridimensional (3D) têm sido desenvolvidos, e as culturas em 3D podem ser uma ferramenta atraente para estudos toxicológicos mostrando a diferenciação celular elevada e a sobrevivência em longo prazo [START_REF] Howes | 3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems[END_REF]MIKHAIL;EETEZADI;ALLEN, 2013;[START_REF] Tung | Highthroughput 3D spheroid culture and drug testing using a 384 hanging drop array[END_REF][START_REF] Vinci | Advances in establishment and analysis of three-dimensional tumor spheroidbased functional assays for target validation and drug evaluation[END_REF]. Modelos de cultura em 3D incluem sistemas de células em várias camadas, cultura celular em uma matriz em 3D, culturas tumorais ex vivo e esferoides tumorais multicelulares (YAMADA; [START_REF] Yamada | Modeling tissue morphogenesis and cancer in 3D[END_REF]. A utilização de geis de colágeno e sistemas multicomponentes mais complexos, tal como Matrigel, tornaram-se cada vez mais populares, uma vez que proporcionam às células uma matriz que representa mais precisamente a matriz extracelular (MEC) [START_REF] Bonnier | Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models[END_REF].

A cultura de células em três dimensões (γD) "possibilita examinar a célula sob um aspecto morfológico e funcional que, de certo modo, se assemelha às condições existentes no tecido in vivo" (REBEδδO, β014). O modelo em γD derivou-se inicialmente da cultura de células em duas dimensões (2D). As células cultivadas em plano bidimensional de plástico ou de vidro são planas e podem aderir e espalhar livremente no plano horizontal, mas não têm suporte para a propagação na dimensão vertical (BAKER; CHEN, 2012). Entretanto, o modelo em 3D permite que as células explorem as três dimensões do espaço tornando favorável a interação entre as células e o ambiente (AMARAL; MACHADO-SANTELLI, 2011).

A cultura de células em 3D, cada vez mais, está sendo reconhecida como um modelo alternativo ao modelo in vivo para melhor compreender a fisiopatologia de células tumorais, e para auxiliar na descoberta de novas terapias anticâncer [START_REF] Howes | 3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems[END_REF]MIKHAIL;EETEZADI;ALLEN, 2013;[START_REF] Saha | Molecular approaches towards development of purified natural products and their structurally known derivatives as efficient anticancer drugs: current trends[END_REF][START_REF] Saha | Molecular approaches towards development of purified natural products and their structurally known derivatives as efficient anticancer drugs: current trends[END_REF]. Além disso, proporcionar um microambiente com adesão e proliferação adequadas permite uma investigação mais precisa da homeostase celular, diferenciação, proliferação e migração [START_REF] Bonnier | Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models[END_REF][START_REF] Kim | Three-dimensional tissue culture models in cancer biology[END_REF].

Como resultado, têm sido desenvolvidos modelos que refletem o microambiente de tumores de sólidos [START_REF] Howes | 3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems[END_REF]. Como representado na Figura 6 a cultura de células cultivada em 3D representa um ambiente intermediário em termos de morfologia celular e fisiologia entre a estrutura simplificada de culturas em monocamada (2D) e a natureza altamente complexa de tumores in vivo (MIKHAIL; EETEZADI; ALLEN, 2013). Entretanto, ensaios baseados em células cultivadas em 2D representam métodos valiosos para identificar rapidamente os compostos citotóxicos e, deste, modo, desempenham cada vez mais um importante papel no processo de descoberta de drogas [START_REF] Gidrol | 2D and 3D cell microarrays in pharmacology[END_REF]. Adicionalmente, os modelos de cultura de células em 3D trazem um valor significativo para auxiliar na descoberta e desenvolvimento de novas terapias anticâncer e funcionam como uma ponte entre as culturas tradicionais em monocamada in vitro e os caros estudos com animais in vivo [START_REF] Howes | 3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems[END_REF][START_REF] Pampaloni | The third dimension bridges the gap between cell culture and live tissue[END_REF][START_REF] Pampaloni | The third dimension bridges the gap between cell culture and live tissue[END_REF][START_REF] Pampaloni | The third dimension bridges the gap between cell culture and live tissue[END_REF][START_REF] Weigelt | Unraveling the microenvironmental influences on the normal mammary gland and breast cancer[END_REF][START_REF] Weigelt | Unraveling the microenvironmental influences on the normal mammary gland and breast cancer[END_REF][START_REF] Yamada | Modeling tissue morphogenesis and cancer in 3D[END_REF][START_REF] Yamada | Modeling tissue morphogenesis and cancer in 3D[END_REF]. Modelos animais são representantes mais precisos de ambiente tumoral, entretanto, são consideravelmente menos suscetíveis ao rastreio em grande escala (GODUGU et al., 2013).

Ensaios de citotoxicidade in vitro

Os testes de citotoxicidade in vitro em cultura de células são importantes para verificar a toxicidade de novos compostos, principalmente no estudo de sua aplicabilidade como agente terapêutico e, pelo menos durante a etapa de triagem, têm reduzido significativamente o número de testes in vivo em animais [START_REF] Hamburger | Bioactivity in plants: the link between phytochemistry and medicine[END_REF][START_REF] Hamburger | Bioactivity in plants: the link between phytochemistry and medicine[END_REF][START_REF] Melo | Violacein cytotoxicity and induction of apoptosis in V79 cells[END_REF].

No desenvolvimento de drogas anticâncer, um painel de linhagens celulares neoplásicas humanas é amplamente utilizado para avaliar os potenciais citotóxicos in vitro de um composto, sendo seus efeitos determinados por diferentes parâmetros: alterações na morfologia celular, viabilidade celular (utilizando corantes como o azul de tripan e a eosina), ensaios clonogênicos, ensaio de incorporação do vermelho neutro, medida de incorporação de nucleotídeos radioativos, marcadores de proliferação celular (Bromodeoxiuridina -BrdU) ou ensaios colorimétricos (MACIEL; PINTO;VEIGA, 2002;MONKS et al., 1991;[START_REF] Sumantran | Cellular chemosensitivity assays: an overview[END_REF].

Os métodos colorimétricos in vitro têm sido amplamente utilizados para a triagem de compostos com atividade anticâncer, pois permitem a quantificação fácil e rápida da resposta celular (SKEHAN et al., 1990). Ensaios que medem a atividade metabólica são adequados para a análise da viabilidade celular, proliferação e citotoxicidade (BERRIDGE; HERST; [START_REF] Berridge | Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction[END_REF][START_REF] Hasen | A cellular viability assay to monitor drug toxicity[END_REF][START_REF] Hasen | A cellular viability assay to monitor drug toxicity[END_REF][START_REF] Tsukatani | Colorimetric microbial viability assay based on reduction of water soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances[END_REF][START_REF] Vellonen | Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity[END_REF][START_REF] Vellonen | Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity[END_REF][START_REF] Vellonen | Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity[END_REF][START_REF] Wang | An improved 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells[END_REF]WANG;[START_REF] Wang | Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO 2[END_REF]WICKLIFFE, 2012). Uma variedade de sais de tetrazólio tem sido MTT em cristais de formazano (MACIEL et al., 2002). Após a adição de dimetilsulfóxido (DMSO), os cristais são solubilizados e quantificados em espectrofotômetro [START_REF] Fisichella | Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes[END_REF][START_REF] Spinner | MTT growth assays in ovarian cancer[END_REF] [START_REF] Houghton | The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity[END_REF]SKEHAN et al., 1990;[START_REF] Vichai | colorimetric assay for citotoxicity screening[END_REF][START_REF] Vichai | colorimetric assay for citotoxicity screening[END_REF]. As duas principais vias de sinalização envolvidas no processo de apoptose em células animais são chamadas de via extrínseca (citoplasmática) e via intrínseca (mitocondrial), e são ativadas, respectivamente, por receptores de morte na superfície celular e da mitocôndria [START_REF] Hengartner | The biochemistry of apoptosis[END_REF][START_REF] Zhao | Antitumor activity of Portulaca oleracea L. polysaccharides against cervical carcinoma in vitro and in vivo[END_REF]. Ambas as vias apoptóticas levam à ativação de caspases (cysteine-dependent aspartate-specific proteases), denominadas de caspases iniciadoras e caspases executoras (ou efetoras), tais como caspases -3, -6 e -7 responsáveis pela execução de eventos intracelulares que resultarão na morte celular por apoptose [START_REF] Gupta | The mitochondrial death pathway: a promising therapeutic target in diseases[END_REF][START_REF] Hajra | Apoptosome dysfunction in human cancer[END_REF][START_REF] Hajra | Apoptosome dysfunction in human cancer[END_REF]ZHANG;[START_REF] Zhang | Involvent of proapoptotic Bcl-2 family members in parthenolide induced mitochondrial dysfunction and apoptosis[END_REF][START_REF] Zhang | Involvent of proapoptotic Bcl-2 family members in parthenolide induced mitochondrial dysfunction and apoptosis[END_REF]) (Figura 9). 

Via intrínseca

A via intrínseca pode ser desencadeada por sinais de "stress" intracelulares, incluindo danos no DNA, o estresse oxidativo ou ativação de oncogenes que conduzem a expressão de vários genes mitocondriais e consequente liberação do citocromo c (um componente da cadeia de transporte de elétrons mitocondriais), do espaço intermembrana mitocondrial para o citosol (BRUIN; MEDEMA, 2008;[START_REF] Galluzzi | Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on cell death 2012[END_REF][START_REF] Zhao | Antitumor activity of Portulaca oleracea L. polysaccharides against cervical carcinoma in vitro and in vivo[END_REF].

A propagação do sinal apoptótico é regulada pelos membros da família de proteínas Bcl-2, o qual consiste em proteínas pró e antiapoptóticas (BRUIN; MEDEMA, 2008). Algumas proteínas Bcl-2 promovem a apoptose pelo aumento da liberação de proteínas próapoptóticas, enquanto as proteínas antiapoptóticas bloqueiam essa liberação e, consequentemente, inibem a apoptose. O balanço entre as atividades dessas duas classes de proteínas Bcl-2 é importante para determinar se células de mamíferos vivem ou morrem pela via intrínseca da apoptose [START_REF] Alberts | Biologia Molecular da Célula[END_REF].

Proteínas Bcl-2 proapoptóticas consistem em duas subfamílias -as proteínas BH123 e as proteínas BH3-apenas (como Bid, Bim, Bad, Noxa e Puma). As principais proteínas BH123 são Bax e Bak [START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Certo | Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members[END_REF]. Na presença de um estímulo apoptótico, proteínas BH3-apenas são ativadas e inibem a função das proteínas Bcl-2 antiapoptóticas (tais como Bcl-2, Bcl-xL, Bcl-w e Mcl-1), permindo assim, a ativação de proteínas próapoptóticas Bax e/ou Bak (BRUIN;MEDEMA, 2008;[START_REF] Certo | Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members[END_REF]CZABOTAR et al., 2014).

As proteínas próapoptóticas Bax e Bak são essenciais reguladores principalmente no controle da liberação do citocromo c e são conhecidas por induzir a permeabilização externa da mitocôndria através da formação de poros por oligomerização [START_REF] Kuwana | and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane[END_REF][START_REF] Wei | Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death[END_REF]. Quando liberado no citosol, o citocromo c liga-se à proteína adaptadora de ativação da procaspase Apaf1 e conduz a formação de um complexo de subunidades múltiplas, conhecido como apoptossomo (PETROS; OLEJNICZAK; [START_REF] Petros | Structural biology of the Bcl-2 family of proteins[END_REF][START_REF] Rupnarain | Colon cancer: genetics and apoptotic events[END_REF].

As proteínas Apaf1 no apoptossomo recrutam então proteínas procaspases iniciadoras (prócaspase-9), as quais são ativadas por proximidade ao apoptossomo [START_REF] Alberts | Biologia Molecular da Célula[END_REF]CZABOTAR et al., 2014). Quando ativada, a prócaspase-9 ativa outras caspases executoras em cascata, tais como caspase-3 para induzir apoptose [START_REF] Certo | Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members[END_REF][START_REF] Zhao | Antitumor activity of Portulaca oleracea L. polysaccharides against cervical carcinoma in vitro and in vivo[END_REF].

Juntamente com a liberação do citocromo c, a proteína SMAC (second mitochondria derived activator of caspases) é liberada do espaço intermembrana mitocondrial para o citosol, ligando-se às proteínas inibidoras da apoptose ou IAPs (Inhibitor of Apoptosis Protein) impedindo, desse modo, que estas exerçam seus papeis antiapoptóticos através da capacidade de inibir a atividade da caspase iniciadora -9 e das caspases efetoras -3 e -7 e portanto, permitindo a progressão da apoptose (CZABOTAR et al., 2014;[START_REF] Reuter | Modulation of anti-apoptotic and survival pathways by curcumin as a strategy to induce apoptosis in cancer cells[END_REF].

Via extrínseca

A via extrínseca desempenha um papel fundamental na manutenção da homeostase dos tecidos, especialmente no sistema imune. Esta via é ativada através da ligação das proteínas de sinalização extracelular aos receptores de morte (DR, Death receptor) presentes na superfície da célula [START_REF] Vermeulen | Apoptosis: mechanisms and relevance in cancer[END_REF].

Receptores de morte são proteínas transmembranas que contem um domínio extracelular de ligação ao ligante, um domínio transmembrana único e um domínio de morte intracelular, o qual é essencial para amplificar o sinal apoptótico. Os receptores são ativados pelos seus ligantes e pertencem à família de receptores do fator de necrose tumoral (TNF, tumor necrosis factor), da qual fazem parte, um receptor para o próprio TNF e o receptor de morte Fas [START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Krueger | Flice-inhibitory proteins: regulators of death receptor-mediated apoptosis[END_REF].

Quando ativados pela ligação do ligante Fas, os domínios de morte nas caudas citosólicas dos receptores de morte Fas recrutam proteínas adaptadoras intracelulares, as quais por sua vez induzem a ativação de caspases iniciadoras (como por exemplo, caspase-8 e caspase-10) e formação do complexo de sinalização indutor de morte (DISC, death-inducing signaling complex). Quando ativadas, as capases iniciadoras ativam caspases executoras para induzirem apoptose [START_REF] Alberts | Biologia Molecular da Célula[END_REF]BRUIN;MEDEMA, 2008). Além disso, em algumas células, como por exemplo, os hepatócitos (tipo II), após formação do complexo DISC, a caspase-8 cliva a proteína pró-apoptótica BID presente no citosol gerando um fragmento truncado dessa proteína (tBid), que por sua vez amplifica o sinal apoptótico por meio da via mitocondrial (CZABOTAR et al., 2014;[START_REF] Gupta | The mitochondrial death pathway: a promising therapeutic target in diseases[END_REF].

Esta ativação é controlada pela proteína bloqueadora como c-FLIP (cellular FLICE inhibitory proteins) (BRUIN; MEDEMA, 2008;[START_REF] Safa | Cellular FLICE-Like Inhibitory Protein (C-FLIP): a novel target for cancer therapy[END_REF][START_REF] Safa | Cellular FLICE-Like Inhibitory Protein (C-FLIP): a novel target for cancer therapy[END_REF]WU, 2008). Essas proteínas se assemelham as caspases iniciadoras e têm a capacidade de competir com as caspases-8 e caspases-10 pelos sítios de ligação no complexo DISC, impedindo a ativação das caspases iniciadoras [START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Krueger | Flice-inhibitory proteins: regulators of death receptor-mediated apoptosis[END_REF][START_REF] Safa | Cellular FLICE-Like Inhibitory Protein (C-FLIP): a novel target for cancer therapy[END_REF][START_REF] Safa | Cellular FLICE-Like Inhibitory Protein (C-FLIP): a novel target for cancer therapy[END_REF]WU, 2008). Esse mecanismo é essencial na regulação da via extrínisica da apoptose e ajuda a impedir a sua ativação inapropriada [START_REF] Alberts | Biologia Molecular da Célula[END_REF].

Ciclo celular

O ciclo celular representa uma série de eventos celulares sequenciais que permitem que a célula cresça e se prolifere. Em eucariotos, o ciclo celular é dividido em quatro fases sequenciais: G1 (G=intervalo 1, do inglês gap), S (S=síntese de DNA), G2 (G=intervalo 2) e M (M=mitose). As fases G1, S e G2 em conjunto são chamadas de interfase [START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Schwartz | Targeting the cell cycle: a new approach to cancer therapy[END_REF][START_REF] Schwartz | Targeting the cell cycle: a new approach to cancer therapy[END_REF].

A fase G1 caracteriza-se pela síntese de proteínas reguladoras necessárias para a replicação do DNA. Quando a fase G1 é completada, a célula entra na fase S onde o DNA é duplicado. No final desta etapa a célula atinge a fase G2, durante a qual as proteínas do fuso mitótico e outros componentes celulares necessários para a mitose são sintetizados (COOPER; [START_REF] Cooper | A Célula: uma abordagem molecular[END_REF]. A mitose compreende dois eventos principais: a divisão nuclear, ou mitose, durante a qual ocorre a duplicação dos cromossomos e a sua distribuição aos núcleos-filhos; e a divisão citoplasmática, ou citocinese, que corresponde à divisão da célula em duas [START_REF] Alberts | Biologia Molecular da Célula[END_REF].

Na ausência de proliferação, as células entram em estado fisiológico de quiescência, denominado de G0 [START_REF] Robbins | Patologia: bases patológicas das doenças[END_REF].

A progressão apropriada através do ciclo celular é um processo altamente regulado em eucariotos por mecanismos denominados de pontos de verificação (checkpoints) [START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Khodjakov | The nature of cell-cycle checkpoints: facts and fallacies[END_REF][START_REF] Khodjakov | The nature of cell-cycle checkpoints: facts and fallacies[END_REF]. Em cada ponto de verificação, numerosas proteínas envolvem-se em uma série de reações O sistema de controle responde a vários sinais intracelulares e extracelulares e interrompe o ciclo quando a célula encontra condições ambientais ou intracelulares desfavoráveis ou a célula falha em completar um processo essencial do ciclo celular como, por exemplo, a replicação do DNA [START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Malumbres | Cell cycle, CDKs and cancer: a changing paradigm[END_REF][START_REF] Malumbres | Cell cycle, CDKs and cancer: a changing paradigm[END_REF]. Nesse momento, ocorre a transcrição de genes que facilitam o processo de reparo [START_REF] Elledge | Cell cycle checkpoints: preventing an identity crisis[END_REF].

Controle da progressão do ciclo celular

Das muitas proteínas envolvidas na regulação da progressão do ciclo celular, as cinases dependentes de ciclina (CDK, Cyclin Dependente Kinases) estão entre as mais importantes. CDKs são enzimas multifuncionais que requerem a associação com uma subunidade reguladora, a ciclina, para a sua ativação (HOCHEGGER; TAKEDA; [START_REF] Hochegger | Cyclin-dependent kinases and cell-cycle transitions: does one fit all?[END_REF][START_REF] Malumbres | Cell cycle, CDKs and cancer: a changing paradigm[END_REF][START_REF] Malumbres | Cell cycle, CDKs and cancer: a changing paradigm[END_REF][START_REF] Schwartz | Targeting the cell cycle: a new approach to cancer therapy[END_REF][START_REF] Schwartz | Targeting the cell cycle: a new approach to cancer therapy[END_REF]. O complexo ciclina-CDK é regulado tanto positiva quanto negativamente por fosforilação [START_REF] Elledge | Cell cycle checkpoints: preventing an identity crisis[END_REF].

As CDKs em associação com as ciclinas tendem a atuar em etapas específicas do ciclo celular. As CDKs existem em quantidades semelhantes ao longo das fases do ciclo, enquanto que a expressão das diferentes ciclinas varia em função da fase do ciclo celular, e a progressão do ciclo é dependente da síntese de Em eucariotos existem três classes de ciclinas, cada uma definida pelo estágio do ciclo celular no qual se ligam às Cdks e em que funcionam.

1. As G1/S-ciclinas ativam Cdks no final de G1 e, com isso, ajudam a desencadear a progressão ao Início, resultando no comprometimento à entrada no ciclo celular. Seus níveis caem na fase S. 2. As S-ciclinas se ligam a Cdks logo após a progressão ao Início e ajudam estimular a duplicação dos cromossomos. Os níveis das S-ciclinas permanecem elevados até a mitose, e essas ciclinas também contribuem ao controle de alguns eventos mitóticos iniciais. Ao final da mitose, vários mecanismos são responsáveis por suprimir a atividade das Cdks, resultando em um período estável de G1 [START_REF] Alberts | Biologia Molecular da Célula[END_REF]. Além disso, as Cdks são ainda reguladas por ligação aos inibidores de CDKs (CKIs), que modificam a sua especificidade, bloqueando, desse modo, sua atividade de cinase [START_REF] Elledge | Cell cycle checkpoints: preventing an identity crisis[END_REF].

Objetivo geral

Avaliar a atividade citotóxica de extratos, frações e da substância isolada da espécie Croton urucurana Baillon (Euphorbiaceae) -"sangra d'água" e investigar o mecanismo de ação de morte celular in vitro para a substância isolada.

Objetivos específicos

 Avaliar a citotoxicidade in vitro de extratos da casca do caule e do látex em seis linhagens de células neoplásicas humanas;

 Selecionar o extrato que apresentar resultados significativos na triagem inicial e submetê-lo ao estudo fitoquímico guiado pela atividade citotóxica frente a seis linhagens de células neoplásicas humanas, a fim de isolar e identificar a substância ativa presente no extrato de interesse;  Avaliar a seletividade da substância isolada em uma linhagem de células não neoplásicas;

 Avaliar os mecanismos celulares in vitro responsáveis pela citotoxicidade da substância isolada considerando seu potencial apoptótico e influência sobre a progressão do ciclo celular em uma linhagem de hepatocarcinoma celular humano (Huh-7), bem como, os efeitos desta substância sobre a migração e viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D. The genus Croton (Euphorbiaceae) comprises about 1300 species of herbs, shrubs, and trees found in tropical and subtropical regions. 1,2 Some of these species have red-colored latex, popularly associated with having therapeutic properties, 3,4 a feature that may have led to its popular names of "dragon's blood", "sangre de drago", and, in Brazil, "sangra-d'água". 5 Phytochemical studies from the latex of some Euphorbiaceae species have led to the isolation of a large number of biologically active constituents, namely, caryophyllaceae-type cyclopeptides. 6,7 This class of compounds was recently redefined as orbitides 8,9 and has attracted much attention due to their important biological activities, such as cytotoxic activity against several human cancer cell lines, 6,7 thus making them a class of promising therapeutic molecules.

Cytotoxic

Croton urucurana Baillon, one of the "sangra-d'água", exhibits a wide range of pharmacological and biological properties. 5,10-15 This plant is commonly found in Mato Grosso do Sul State (midwestern Brazil), where its latex is popularly used to treat several types of cancer. Despite the ethnopharmacological significance of the species, no phytochemical or cytotoxic studies of its latex have been published.

As part of an ongoing research program to discover potential anticancer compounds from plants of midwestern Brazil (Cerrado and Pantanal biomes), this article reports the isolation of a novel orbitide (1) from the latex of a specimen of C. urucurana, as well as the determination of its cytotoxicity against a panel of human cancer cell lines using the sulforhodamine B (SRB) assay.

RESULTS AND DISCUSSION

The phytochemical investigation of C. urucurana was guided by the cytotoxic activity (detected using the SRB assay) exhibited against 786-0 (kidney carcinoma), HT-29 (colon carcinoma), NCI-ADR/RES (ovary adenocarcinoma, multidrug-resistance phenotype), MCF-7 (breast adenocarcinoma), PC-03 (prostate carcinoma), and Hep-G2 (hepatocellular carcinoma).

We initially examined the EtOH extract obtained from bark, which proved inactive (GI 50 > 250 µg mL -1 ), and also the latex, which proved cytotoxic against all the cancer cell lines investigated (Table 1). The latex was therefore selected for further cytotoxicity-guided isolation and phase-partitioned between MeOH-H 2 O and hexane and subsequently between MeOH-H 2 O and EtOAc. Of the six fractions partitioned from the bioactive EtOAc phase, fraction 2 (F2) was the most active against 786-0 (GI 50 = 5.42 µg mL -1 ) and NCI-ADR/RES (GI 50 = 3.93 µg mL -1 ) cells (Table 1). Further fractionation of F2 led to isolation and structure elucidation of cytotoxic compound 1, its major constituent. 2).

2D NMR experiments not only corroborated the foregoing inference, but also led to identification of nine amino acid residues: three glycines (G a , G b , G c ), two leucines (L a , L b ), one phenylalanine (F), one asparagine (N), one alanine (A), and one serine (S) (Table 2).

The absolute configurations of the A, F, L, and S residues were identified as L(S) using εarfey's method. 16 On the basis of NOE data, the configuration of the N residue was determined to be L(S). This finding is in agreement with studies showing that all cyclic peptides isolated from Croton species, 17,18 and likewise from other Euphorbiaceae, 19-31 consist exclusively of L amino acids.

On the basis of correlations observed in the COSY 1 H-1 H and NOESY spectra, the cyclic nature of this peptide and its amino acid residue sequence were identified (Figure 1). Noteworthy in the former experiment were the correlations observed between the amino acid Hα and its NH group, which permitted all hydrogens of each amino acid residue to be assigned. Likewise, the NOESY correlations among NH groups facilitated definition of the other amino acids immediately preceding or succeeding each residue. A detailed analysis of these data (Table 2) revealed the amino acid sequence in 1 as being (cyclo)-G a SL a G b AFG c NL b -. Characterization of 1 was also confirmed by its HRESIMS/MS data, which showed fragments consistent with the proposed sequence of amino acid residues (Table 3). The fragmentations of cyclic peptides are dominated by the b x -y z pathway, in which ring opening takes place via oxazolone formation, resulting in linear peptide ions having a free N-terminus and an oxazolone ring at the C-terminus. 32 These linear ions can give rise to lower b fragments by the b x -b x-1 pathway, in addition to undergoing other possible fragmentations, such as CO loss via the b x -a x pathway, subsequent losses of amino acid residues by the b x -y z pathway, and common neutral losses (H 2 O, NH 3 ). 32,33 Although no data are available on the loss of ammonia from the N-terminal amino group of a linear ion, this loss does occur from the side chain of asparagine, glutamine, aspartic acid, and arginine residues. 32 On the basis of its skeleton, this compound can be classified as a caryophyllaceae-type cyclopeptide 6 recently redefined as an orbitide. 8,9 According to Shim, Young, Arnison, Gilding, and Reaney, 9 compound 1 can be designated as

[19-NαC]-crourorb A1.
A range of biological properties have been reported for orbitides, including immunomodulatory, 19,20 antimalarial, 21,24,28,29,31 antifungal, 23 and antibacterial activities. 35 In recent years, considerable attention has been devoted to the anticancer activity of natural cyclic peptides, 6,27,36-38 which have been considered a new class of therapeutic molecules, due to their chemical versatility and specificity for biological targets. 39 Evaluated against six human cancer cell lines, compound 1 proved almost as potent against NCI-ADR/RES cells (GI 50 = 4.8 µM) as doxorubicin, the positive control (GI 50 = 5.1 µM) (Table 1), suggesting that 1 is the component responsible for toxicity against this cell line in the preceding fraction F2. When tested against 786-0 cells, however, F2 exhibited lower GI 50 value than 1, suggesting that the cytotoxic activity of this fraction against this cell line stems from other active minor compounds or from synergistic interactions lost during fractionation. Evaluated against NIH/3T3 nontumor cells, 1 remained inactive up to the highest concentration tested (GI 50 > 305.8 µM), thus demonstrating selective inhibition against NCI-ADR/RES cells. In addition, doxorubicin proved cytotoxic against NIH/3T3 cells (GI 50 = 4.0 µM), emphasizing a possible advantage in the cytotoxic potential of 1, which would have a reduced adverse effect in normal tissues. Orbitides have been reported to have potent cytotoxic activity and may exhibit differences in responsiveness/sensitivity to different cancer cells lines. 6,7 Nevertheless, further studies are necessary to determine the cytotoxic effects on nontumor cells and the limitations of this class of compounds.

Before the present study, the first of its kind reporting the isolation of a cyclopeptide from C. urucurana latex, this class of compounds had been identified in other Euphorbiaceae. 19-31 Cyclopeptides had also been found in C. gossypifolius 17 and C. sparsiflorus, 18 but their cytotoxic activities against cancer cell lines were not evaluated. These cyclopeptides are characterized by the presence of nine amino acid residues, of which phenylalanine, asparagine, alanine, glycine, and leucine occur in both chains and three glycine residues occupy the same positions. positive HRESIMS/MS data, see Table 3.

Marfey's Analysis of the Absolute Configuration of Amino Acid 16,40 Compound 1 (1 mg) was hydrolyzed in 1 mL of 6 N HCl at 95C for 16 h. The hydrolysate was evaporated to dryness under a N 2 stream to remove traces of HCl and redissolved in 900 µL of acetone and 1 M NaHCO 3 (20 µL). A solution of N-α- L-Leu (13.1 and 9.4), D-Leu (18.9 and 9.4), L-Phe (12.5 and 9.4), D-Phe (16.6 and 9.4), L-Ser (8.8 and 15.1), and D-Ser (9.2 and 15.0). The retention times (min) observed for L-FDAA derivatives of acid hydrolysates of compound 1 and for the residual εarfey's reagent were as followsμ δ-Ala (7.4 and 9.7, respectively), L-Leu (13.0 and 9.7), L-Phe (12.4 and 9.7), and L-Ser (8.6 and 15.3).

In Vitro Cytotoxicity Assay

The cytotoxic activities of the extract, phases, fractions, and compound nontumoral cells were purchased from the Rio de Janeiro Cell Bank.

Briefly, the cells were seeded in 96-well plates at a density of 7.5 × 10 3 cells/well and each sample was tested in triplicate at four concentrations (0.25, 2.5, 25, and 250 µg mL -1 ) at 37 C and 5% of CO 2 for 48 h. The samples were dissolved in DMSO, aliquoted, and stored at -20 C. Final DMSO concentration did not affect cell viability (0.25%). Doxorubicin hydrochloride (2 mg/mL, Libbs) was used as the positive control. Growth inhibition by 50% (GI 50 ) was determined by nonlinear regression analysis using Origin 6.0 software (OriginLab). 42,43 Samples were considered inactive when GI 50 values exceeded 250 µg mL -1 .

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00724.

1D and 2D NMR, HRESIMS, HRESIMS/MS, UV, and IR spectra of compound 1 (PDF). 

Introduction

Medicinal plants play an important role in discovery novel bioactive compounds for drug development, especially for anticancer therapy [START_REF] Pan | Discovery of new anticancer agents from higher plants[END_REF]Cragg and Newman, 2013). Recently, plant-derived cyclic peptides have emerged as therapeutically potent anticancer biomolecules (Yue et al., 2011;Fang et al., 2013;Mishra et al. 2014).

Studies have shown that cyclic peptides from plants exhibit high cytotoxic activity through cell cycle arrest and inducing apoptosis in cancer cell lines [START_REF] Ma | Peptides from Plants: A New Source for Antitumor Drug Research[END_REF]Mishra et al. 2014;Yeu et al., 2011). In addition, they have also exhibits a wide range of pharmacological properties including immunomodulatory, antimalarial, antifungal, antibacterial, antiplasmodial, insecticidal and sedative (Tan and Zhou, 2006).

Cyclic peptides have been discovered from medicinal plants species of the families Compositae, Caryophyllaceae, Labiatae, Linaceae, Annonaceae, Amaranthaceae, Rubiaceae and Euphorbiaceae (Tan and Zhou, 2006). Among Euphorbiaceae species it is highlighted Croton urucurana Baillon, which is common plants found in Mato Grosso do Sul state (midwestern Brazil) and its latex has been used in folk medicine for treating cancer [START_REF] Cândido-Bacani | Cytotoxic orbitide from the latex of Croton urucurana[END_REF].

Based in our research program which aims to the discovery of potential anticancer compounds from plants of midwestern of Brazil (Cerrado and Pantanal biomes), we previously isolated a novel cyclic peptide, designated as [19-NαC]crourorb A1, as major component from Croton urucuana latex and demonstrated its cytotoxic activity against a panel of cancer cell lines [START_REF] Cândido-Bacani | Cytotoxic orbitide from the latex of Croton urucurana[END_REF].

However, the involved underlying mechanisms in the crourorb A1-cytotoxicity against cancer cells remain unknown.

Considering the potential therapeutic of crourorb A1 and due to lack of information on the cellular and molecular mechanisms underlying responsible for crourorb A1-induced cytotoxicity on cancer cell lines, in the present study we investigated the effects of crourorb A1 on the viability, apoptosis, cell cycle and cell migration of Huh-7 cells. Finally, we evaluated the role crourorb A1 on the viability of Huh7 cells in 3D collagen culture.

Materials and Methods

Cell lines and culture

The human hepatocarcinoma cell lines Huh-7D12 was obtained from Health Protection Agency Culture Collections (ECACC, European Collection of Cell Cultures nº 01042712). Cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal calf serum (FCS) and antibiotics (penicillin 100 IU/ml and streptomycin 100 g/ml). The cells were grown in plastic Petri dishes (100 mm, Corning, NY) in humidified atmosphere of 5% CO 2 at 37º C. Crourorb A1 was initially diluted in DMSO, aliquoted and stored at -20ºC. The concentration of DMSO in culture medium was kept below 0.2% (v/v), a concentration known not to affect cell viability.

Materials

3D Collagen Culture

Huh-7 cells were seeded into type I collagen gels (Sigma-Aldrich, St-Quentin-Fallavier, France) diluted with culture medium to obtain a collagen solution of 0.75mg/ml and 1.5mg/ml. The pH of solution was adjusted to 7.4 by NaOH 0.1N and cell suspension was mixed to this solution to give a final cell density of 40.10 3 cells/ml. Collagen gels were then distributed into 96-well plates (100 L) or 48-well plates (250 L) and incubated at 37°C. After polymerization (10 min at 37°C), gels were covered with an equal volume of culture medium.

Cell viability assay

The cell viability of Huh-7 cells was evaluated by colorimetric WST-1 assay (Roche, Germany). The assay is based on the ability of viable cells to cleave the

sulfonated tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5- tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases. Briefly, cells
were seeded in 96-well plates in 2D (8 x 10 3 ) or 3D collagen gels (4 x 10 3 ) and treated with increasing concentrations of crourorb A1 (0.35-200 µg/ml) or 0.2% DMSO as control for 24h, 48h, and 72h (2D) or (10-100 µg/ml) or 0.1% DMSO for 24h after, 6 days of cultures (3D). After treatments, 100 L of the WST-1 reagent solution (10% in culture medium) was added to each well and incubated for 2h at 37°C (2D) or for 1h at 37°C (3D). The absorbance was measured at 430nm on SPECTROstar Nano (BMG Labtech, Champigny sur Marne, France) and percentage of viability was calculated as the absorbance ratio of treated to untreated cells. The concentration that inhibited 50% cell growth (IC 50 ) was determined through non-linear regression analysis.

Necrosis study

Huh-7 cells were plated on 35 mm petri dish and treated with crourorb A1 (36µg/ml) or 0.036% DMSO as control for 24 and 48 h. At the indicated times the medium was removed, and the cells were washed twice with PBS and stained with Hoechst 5 g/ml and Propidium Iodide 5 g/ml at γ7º C for 30 min in the dark. After washing with PBS was added 3 drops fluorescein diacetate (DAF) and the petri dish were covered with coverslips and observed in fluorescent microscopy (ZEISS Axio Scope.A1). The doubled stained nucleus corresponding to necrosis cells were counted and the results are shown as the mean percentages of necrosis cells.

Caspase-3/7 activity

Caspase-3/7 activity was measured using SensoLyte Homogeneous AMC Caspase-3/7 Assay kit (Anaspec, Le-Perray-en-Yvelines, France) following manufacturer's instructions. Briefly, Huh-7 cells were seeded in 96-well plates in 2D at density of 8 x 10 3 cells/well and treated with increasing concentrations of crourorb A1 (10-50 µg/ml) or 0.05% DMSO as control for 24h. At indicated time, cells were lysed on the plate and the caspase-3/7 substrate solution was incubated with the cell lysates for 1h at 37°C. AMC cleavage was monitored by spectrofluorometry (Vmax).

WST-1 assays were performed in parallel to assess the approximate number of viable cells in each well. Cisplatin was used as positive control (20µg/ml).

Cell cycle analysis

Huh-7 cells (3×10 5) were seeded at 6-well culture plates and incubated overnight. The next day, cells were treated with crourorb A1 at 36 or 50 µg/ml or 0.05% DMSO as control for 24 h. Cells were fixed with 70% ice-cold ethanol for 1h and incubated for 30 min at 37º with 0.5U of RNase A (Sigma-Aldrisch). DNA was then stained for 10 min with 50 g/ml of PI in the dark and subjected to flow cytometric analysis using a FC500 flow cytometer (Beckman Coulter, Paris, France).

The percentage of cells in G0/G1, S and G2/M phases was determined using the KALUZA 1.3 analysis software (Beckman Coulter).

Western blot analysis

Huh-7 cells were treated with crourorb A1 at 36µg/ml or 0.036% DMSO as control at different times. Cells were washed with ice-cold PBS, scrapped and frozen at-β0°C. The protein lysates were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% low fat milk in trisbuffer saline (TBS) (65mM Tris pH 7.4, 150mM NaCl) for 1h at room temperature, incubated with primary antibodies overnight at 4°C and then incubated with appropriate secondary antibodies linked to horseradish peroxidase in 5% low fat milk in TBS for 1h at room temperature. Proteins were visualized with Immobilon Western Chemiluminescent HRP substrate (VWR) and scanned with the Fujifilm LAS-3000 imager (Fujifilm, Tokyo, Japan). Densitometric analyses of the bands were carried out with the MultiGauge software (Fujifilm).

Wound healing assay

Huh-7 cells were seeded in 12-well plates at density of 1x10 5 cells/well and grown until 90% confluence at 37ºC. The culture medium was discarded and scratchwound was performed using a sterile 200 µl pipet tip and cells were washed twice with PBS to remove the scratched cells. Huh-7 cells were cultured in the absence or presence of mitomycin C (1µg/ml) and treated with crourorb A1 (36 µg/ml) or with 0.036% DMSO as control for 24, 48 and 72h after the scratching. The initial wounding and subsequent migration of the cells in the scratched area were photographed in four different fields under microscope (AXIOVERT 40 C Zeiss). The results are presented as percentage width of the gap against the value of the negative control at 0 h of crourorb A1 treatment.

Cells staining

Huh-7 cells (2D) were seeded in 12-well plates and treated with 50µg/ml of crourorb A1 or 0.050% DMSO as control for 24h. The culture medium was removed, and the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 20 min, rewashed, and permeabilized in 0.1% Triton X-100/PBS for 10 min, and blocked in PBS 2%/BSA for 1h at room temperature. The slides were stained with Phalloidin-Fluoprobes 457H (Interchim) 66nmol/L and Hoechst 5µg/ml and observed by fluorescence microscopy (Nikon Eclipse Ni).

Growth kinetics of the spheroids (3D) were followed after 4 days of cultures, for 48h after treatment with crourorb A1 (62µg/ml) or 0.062% DMSO as control and morphological changes were assessed by fluorescent dye labeling. The culture medium was removed, and the cells were washed twice with PBS, fixed in 4% paraformaldehyde for 20 min, then rewashed and incubated with PBS containing 50 ε of RS1λ for β0 minutes and observed with Leica TCS SP2 confocal scanning head coupled to a DMIRB inverted microscope (Leica Microsystems, Mannheim, Germany).

In addition, collagen gels were deshydrated with successive increasing concentration of alcohol and Xylene baths before being impregnated with paraffin using EXCELSIOR ES tools (Thermo Scientific, Waltham, USA) overnight. After impregnation, gels were include in paraffin and bloc 4-m-thick sections were cut and stained with hematoxylin and eosin.

Statistical analysis

Results are expressed as mean ± standard deviation. Data were analyzed with two-tailed Student's t-test. Differences were considered significant when p<0.05(*), p<0.01(**), p<0.001(***). All experiments were performed at least three times.

Results

Effect of crourorb A1 on hepatic cells viability

In vitro cytotoxicity of the crourorb A1 against Huh-7 cells was assessed by WST-1 assay. Crourorb A1 decreased the cell viability of Huh-7 at dose-and time dependent manner and the cell viability decreased with increasing concentration and incubation duration (β4, 48 and 7βh). As shown in Figure βA, 50, 100 and β00 g/ml of crourorb A1 led to a great inhibition in cell viability already after 24 h of treatment (p < 0.01). The effects were maintained and more pronounced after 48 and 72h of treatment. With lower crourorb A1 concentrations (0.γ5, γ and 1β.5 g/ml) no or little influence on the cell viability was observed.

Crourorb A1 was also tested in a wide range of concentrations, from 10 g/ml to 70 g/ml for β4h. The concentrations of γ0, 40, 50 and 70 g/ml reduced the cell viability to 80%, 20%, 13% and 8% after 24h treatment, respectively (p < 0.01) (Figure 2 B).

The dose-response curve was used to determine crourorb A1 concentrations required to inhibit the growth of cancer cells by 50% (IC 50 ). The IC 50 values at 24, 48 and 72 h were estimated at 35.75±1.00, 30.41±2.14 and 20.07±3.40 g/ml, respectively (Table 1). IC 50 value for 24h of the treatment with crourorb A1 was used as an effective concentration for subsequent assays.

Crourorb A1 induces apoptosis and G2/M cell cycle phase arrest

Morphological alterations of nucleus after treatment with 50 µg/ml of crourorb A1 for 24h exhibited apoptotic bodies after Hoechst staining, as compared to control cells (Figure 3 A). Figure 3 B shows the percentage of necrotic cells after 24h and 48h of treatment with crourorb A1 at the IC 50 (36µg/ml). As shown in Figure 3B there were no significant differences compared to the DMSO control and only basal levels of necrotic cells can be observed.

To evaluate if the inhibition of the cell viability in response to crourorb A1 was due to the induction of apoptosis, caspase 3/7 activity was measured. Treatment of Huh-7 cells with crourorb A1 at γ0, 40 and 50 g/ml for β4 h resulted in significant increased caspase 3/7 activity compared to control cells (Figure 3C) (p<0.05; p<0.001). At higher concentrations (70 µg/ml), crourorb A1 did not show significant caspase 3/7 activity increasing because of its high cytotoxicity leading to considerable cell death (data not shown).

Since the pro-apoptotic Bcl-2 family is the principal pathway involved in apoptosis, the levels of Bak, Bad, Puma, Bax, Bim, Bid and the cleaved-caspase-3

protein were analyzed by Western blot after 10, 24 and 48 h of treatment with crourorb A1. The results showed that the levels of all the pro-apoptotic proteins analysed increased in crourorb A1-treated cells. Similar results were also observed in the levels of cleaved caspase-3 in crourorb A1-treated cells after 24 and 48h of treatment (Figure 3D).

To determine the influence of crourorb A1 on cell cycle progression, flow cytometry analysis was performed and G0/G1, S and G2/M cell cycle phases were detected by PI staining. Our results showed a dose-dependent increase in G2/M phase in cells treated with 36 and 50 µg/ml of crourorb A1 for 24h (Figure 4 In order to study role of the phospho-ERK1/2, phospho-AKT and phospho-JNK in crourorb A1 survival/cytotoxicity, we analysed the effect of these pathways inhibitions on cell viability by WST-1 assay. As shown in Figure 5 B, after 24 h of treatment there was a blockage of crourorb A1 toxicity in cells treated with the JNK inhibitor. In contrast, no significant changes were detected after treatment with the MEK inhibitor U0126 or Wortmannin, a PI3K/AKT inhibitor (p>0.05). These results indicated that the JNK pathway is involved in the crourorb A1-induced cell mortality (Figure 5 C).

Then, the effects of crourorb A1 in cell motility, was examined using a monolayer wound healing assay. As shown in Figure 6, wound closure was markedly delayed in crourorb A1-treated cells compared to control DMSO-treated cells, whether the cells are cultured in presence or absence of mitomycin C (Figures 6 A-C). These results revealed that crourorb A1 could inhibit the migration of Huh-7 cells in a time-dependent manner (p<0.05) independently to its cell cycle inhibitory effect.

Crourorb A1 inhibits viability of Huh-7 cells in 3D cultures

Mechanical forces could greatly influence the cells viability of hepatoma cells.

In vitro, 3D collagen gels provide valuable flexible model for studying cells in a 3D

environment and the response to variations of stiffness. This approach allowed us to establish that rigidity could influence the phenotype of hepatoma cells embedded in the matrix [START_REF] Bomo | Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment[END_REF]. In this work, we show that 3D environment in collagen gels can also influence the response of the cells to crourorb A1 and an increase of cell viability in 3D gels was observed following treatments with 50 and 100 g/ml of crourorb-A1 (Figure 7). The IC 50 values at β4h were 6β.10±4.70 g/ml and 6β.5β±γ.1β g/ml in collagen gels at 0.75mg/ml (1Pa) and 1.5mg/ml (γ.5Pa), respectively (Table 2), which are 1.8-fold higher than in 2D culture.

In order to observe at the cluster organization of crourorb A1-treated Huh7 corroborate with the results reported in our previous study [START_REF] Cândido-Bacani | Cytotoxic orbitide from the latex of Croton urucurana[END_REF] which showed that concentrations ranging from βλ to 41 g/ml inhibited by 50% the viability of HT-29 (colon carcinoma), MCF-7 (breast adenocarcinoma), Hep-G2 (hepatocellular carcinoma) and PC-03 (prostate carcinoma) human cancer cells. In contrast, crourorb A1 presented potent cytotoxic activity against adryamycin-resistant ovarian (NCI-ADR/RES) cancer cells (IC 50 =3.98 µg/ml) and moderate cytotoxicity against human kidney (786-0) cancer cells (IC 50 =18.69 µg/ml). Also, it was shown that crourorb A1 had no effect on non-tumor cell viability (NIH/3T3 murine fibroblast cells) (IC 50 = >250µg/ml) [START_REF] Cândido-Bacani | Cytotoxic orbitide from the latex of Croton urucurana[END_REF]. These results suggest that crourorb A1 may selectively inhibit the viability of cancer cells with differences in responsiveness/sensitivity depending to the cancer cell lines whereas immortalized cells are not/or less sensitive to crourorb A1.

Since apoptosis have a central role to limit the uncontrolled proliferation of cancer cells [START_REF] Elmore | Apoptosis: A Review of Programmed Cell Death[END_REF][START_REF] Ziegler | Therapeutic targeting of apoptosis pathways in cancer[END_REF] we investigated apoptotic effect of crourorb A1 in the highly proliferative hepatocarcinoma cell line Huh-7. Apoptosis is characterized by characteristics morphological changes which include cell shrinkage, membrane blebbing, DNA fragmentation, chromatin condensation and apoptotic bodies. These changes requiring activation of several signaling cascades which are regulated by various cellular proteins [START_REF] Sprick | The interplay between the Bcl-2 family and death receptor-mediated apoptosis[END_REF][START_REF] Elmore | Apoptosis: A Review of Programmed Cell Death[END_REF]Bruin and Medema, 2008;[START_REF] Estaquier | The mitochondrial pathways of apoptosis[END_REF], including the Bcl-2 family proteins. Indeed, it has been shown that this family included pro-apoptotic (Bax, Bak, Bcl-XS, Bid...) and anti-apoptotic (Bcl-2, Bcl-XL, Mcl-1, A1, Bcl-W...) which play an essential role in this mechanism (Burlacu, 2003;[START_REF] Chipuk | How do Bcl-2 proteins induce mitochondrial outer membrane permeabilization?[END_REF]Song et al., 2013). These proteins regulate the mitochondrial membrane permeability and promote the activation of different classes of caspases (initiators and effectors) leading to apoptotic cell death [START_REF] Chipuk | How do Bcl-2 proteins induce mitochondrial outer membrane permeabilization?[END_REF]Green, 2008, Song et al., 2013;Czabotar et al., 2014). The caspase family plays a crucial role in the implementation of apoptosis and caspases activations appear to be linked to molecular and structural changes that occur during apoptosis. Among the identified caspases, caspase-3 is known to be the major executioner caspase of the apoptotic cell death program [START_REF] Elmore | Apoptosis: A Review of Programmed Cell Death[END_REF]. In this study, we observed by fluorescent dye labeling of Huh-7 that crourorb A1 caused a morphological changes characteristic of apoptosis. Crourorb A1 induce-apoptosis was confirmed by an increase of caspase-3/7 activities in a dose dependent manner, accompanied by the up-regulation of clived-caspase3 protein and expressions of pro-apoptotic proteins such as Bak, Bid, Bax, Puma, Bim, and Bad. Taken together, our data show that crourorb A1-induced cytotoxicity could be strongly mediated through a caspase-3-dependent pathway and pro-apoptotic proteins up-regulation. Apoptotic effect of natural cyclic peptides, such as jasplakinolide was also demonstrated in human leukemia Jurkat T cells by [START_REF] Odaka | Jasplakinolide Induces Apoptosis in Various Transformed Cell Lines by a Caspase-3-Like Protease-Dependent Pathway[END_REF]. Jasplakinolide-induced apoptosis was associated with an increased of caspase-3 activity and Bax levels accompanied by a decrease in the expression of Bcl-2 in Jurkat T cells [START_REF] Odaka | Jasplakinolide Induces Apoptosis in Various Transformed Cell Lines by a Caspase-3-Like Protease-Dependent Pathway[END_REF]. Jasplakinolide was also capable of inducing apoptosis in various murine transformed lines, including T-cell lymphoma EL-4 cells, myeloma SP-2/0 cells, macrophage-like J774.1 cells and fibroblast L cells [START_REF] Odaka | Jasplakinolide Induces Apoptosis in Various Transformed Cell Lines by a Caspase-3-Like Protease-Dependent Pathway[END_REF]. Cozzolino et al. (2005) Additionally, we investigated the effect of crourorb A1 on cell cycle progression. In the present study, we shown that crourorb A1 induced a blockage of cell cycle progression of the Huh-7 cells by accumulation of cells in G2/M phases. In parallel, we observed that crourorb A1 treatment resulted in an increase of CDK1 and cyclin B1 expressions. Since the Cdk1/cyclin B1 complex play an important role in the transition from G2 to M phase [START_REF] Murray | Recycling the Cell Cycle: Review Cyclins Revisited[END_REF]), here we suggest that crourorb A1 could increase the proportion of cells in G2/M phase by blocking cells in late G2

and/or beginning of M phase expression related to an increase of the CDK1 and cyclin B1expression pattern. Indeed, natural cyclic peptides have been reported to inhibit cancer cell growth mediated by cell cycle arrest in the G2/M phase in cancer cell lines (Yeu et al., 2011;[START_REF] Du | A Potent HDAC Inhibitor, 1-Alaninechlamydocin, from a Tolypocladium sp. Induces G2/M Cell Cycle Arrest and Apoptosis in MIA PaCa-2 Cells[END_REF], a feature regarded as an effective strategy for inhibiting cancer cell growth [START_REF] Zhang | Effect of Lycium barbarum polysaccharide on human hepatoma QGY7703 cells: Inhibition of proliferation and induction of apoptosis[END_REF]. Cell migration is an essential step in cancer progression, leading to invasion and metastasis [START_REF] Pasco | Control of melanoma progression by various matrikines from basement membrane macromolecules[END_REF]. In our study, we also demonstrated the effects of crouroub A1 on cell migration of Huh-7cells cultivated in the absence and presence of mitomycin C.

Mitomycin C is an antibiotic highly effective against various types of human cancer, as it is capable to block mitosis and inhibit cancer cell growth. In this regard, it becomes possible to analyze cell migration in the absence of cell proliferation [START_REF] Mao | Genetic Localization and Molecular Characterization of Two Key Genes (mitAB) Required for Biosynthesis of the Antitumor Antibiotic Mitomycin C[END_REF][START_REF] Gen | A novel amplification target, ARHGAP5, promotes cell spreading and migration by negatively regulating RhoA in Huh-7 hepatocellular carcinoma cells[END_REF]. Crourorb A1 treatment was shown to inhibit the migration potential of Huh-7 cells by wound healing analysis both in absence and presence of mitomycin C excluding the possibility that its effect on cell migration could be consequence of the crourorb A1-induced inhibition of cell proliferation.

PI3-AKT, p70S6K and MAPK pathways play a key role in the cell migration, survival/apoptosis and proliferation [START_REF] Chang | Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention[END_REF][START_REF] Le | Paclitaxel induces inactivation of p70 S6 kinase and phosphorylation of Thr421 and Ser424 via multiple signaling pathways in mitosis[END_REF][START_REF] Dhillon | MAP kinase signalling pathways in cancer[END_REF]Zang et al., 2011;[START_REF] Hein | Radiation-induced signaling pathways that promote cancer cell survival (Review)[END_REF]. Thus, we analyzed the phosphorylation state of the MAP kinases p38, ERK, JNK, AKT and p-70S6K in response to crourorb A1. We showed that JNK, ERK1/2 and AKT pathways were activated after crourorb A1 exposure in a time-dependent manner whereas the phosphorylation levels of p38 and p70S6K were not affected as compared to the control cells with solvent (DMSO).

Moreover, we determined that the JNK pathway was necessary for crourorb A1induced cell death as evidenced by protection of crourorb-apoptosis induction by the JNK inhibitor II. On the other hand, although crourorb A1-activated ERK1/2 and AKT pathways, the inhibitors U0126 and Wortmanin were not effective in preventing induced cell death, showing that ERK1/2 and AKT activations seem to be not involved in the cytotoxicity of crourorb A1 in Huh-7 cells.

On the basis on the knowledge that 3D cultures better approximate the in vivo environment and can lead to divergent results when compared to 2D cultures (Mikhail et al., 2013;Eicheler et al., 2014), we quantified the viability of 3D cells, in stiff and soft matrix, after crourorb A1 treatment. Huh-7 cells in these matrices exhibit changes in morphology and proliferation. In soft collagen gels (collagen 0.75mg/ml), Huh-7 cells appear well-spread and form monolayer-like structures with flattened shapes while in rigid gels (collagen 1.5mg/ml) cells are non-spread and form rapidly spherical clusters along the culture time [START_REF] Bomo | Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment[END_REF]. In our study, we showed that cells grown in 3D collagen gels exhibited increasing resistance to crourorb A1 when compared to 2D cell culture; however no difference was observed for both 3D culture models. IC 50 values were 1.8-fold higher in both 3D models compared to conventional 2D monolayer cultures. Similar results were obtained for the positive control cisplatin. Moreover, 3D reconstruction of spheroids treated with crourorb A1

showed significant morphological alterations and cell surface decreasing when compared to untreated cells. These data are in accordance with previous studies that showed also a greater resistance to the anti-cancer drug in 3D cultures in comparison to 2D-cultured cells [START_REF] Yip | A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing[END_REF]Godugo et al., 2013;Mikhail et al., 2013;Eicheler et al., 2014;[START_REF] Lauer | Single molecule microscopy in 3D cell cultures and tissues[END_REF]. This resistance to anticancer drugs in γD models has been attributed to several mechanisms, including a "decreased penetration of anticancer drugs, increased pro-survival signaling, and/or upregulation of genes conferring drug resistance" (Godugo et al.,β01γ). In regards the spheroids has the "ability to block the diffusion of the drug to all the cells as cells on the outer layers of the spheroid provide a natural barrier" (Yip and Cho,β01γ). However, the 

RÉSUMÉ

  CÂNDIDO-BACANI, PRISCILA DE MATOS. Un nouveau cyclopeptide cytotoxique extrait du latex de Croton urucurana Baillon (Euphorbiaceae); étude in vitro de son activite cytotoxique. Campo Grande (MS); 2016. [Thèse de doctorat en cotutelle -Programa de Pós-graduação em Saúde e Desenvolvimento na Região Centro-Oeste, Universidade Federal de Mato Grosso do Sul et Ecole doctorale VAS, Université de Rennes 1, Rennes].

22 Figura 1 - 23 Figura 2 - 28 Figura 4 - 29 Figura 5 - 33 Figura 6 - 35 Figura 7 - 37 Figura 8 - 39 Figura 9 -Figure 1 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 1 .Figure 2 .Figure 3 .

 221232284295336357378399112345678123 Figura 1 -Estruturas de algumas drogas anticâncer derivadas de plantas em uso clínico. ....................................................................................................................... 23 Figura 2 -Espécies de Croton, usos tradicionais e efeitos farmacológicos de partes das plantas e alguns compostos importantes isolados ............................................ 27 Figura 3 -A) Árvore de Croton urucurana Baill. B) Detalhe das folhas. C) Detalhe da florescência. ............................................................................................................. 28 Figura 4 -Extração do látex de "sangra d'água" ....................................................... 29 Figura 5 -Bancos fornecedores de linhagens celulares. ........................................... 33 Figura 6 -Cultura de células 3D como intermediária entre cultura de células 2D e modelo animal ........................................................................................................... 35 Figura 7 -Estrutura química dos diferentes sais de tetrazólio e o aceitador de elétrons intermediário, metassulfato de fenazina (PMS) ........................................... 37 Figura 8 -Estrutura da SRB ...................................................................................... 39 Figura 9 -Vias de sinalização envolvidas na apoptose: via mitocondrial e via extrínseca .................................................................................................................. 40

Figure 1 -

 1 Figure 1 -Fracionamento biomonitorado do látex de C. urucurana por meio do ensaio de citotoxicidade com o corante sulforrodamina B ......................................

Figure 2 -LISTANaHCO 3

 23 Figure 2 -Perfil cromatográfico da Fração 2 por CLAE ...........................................

2. 6 . 1 1

 611 Controle da progressão do ciclo celular ........................................................... Objetivo geral ...................................................................................................... 3.2 Objetivos específicos .......................................................................................... ..................................................................................................... REFERÊNCIAS ....................................................................................................... APÊNDICES ..........................................................................................................

Figura 3 -

 3 Figura 3 -A) Árvore de Croton urucurana Baill. B) Detalhe das folhas. C) Detalhe da florescência.

Figura 6 -

 6 Figura 6 -Cultura de células 3D como intermediária entre cultura de células 2D e modelo animal.

  utilizada para medir a atividade metabólica de células viáveis, como o MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (2,3-bis(2-methoxy-4nitro-5-sulphophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt) e WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt) (BERRIDGE; HERST; TAN, 2005; YIN et al., 2013) (Figura 7). Esses compostos são classificados em duas categorias básicas: 1) MTT que é carregado positivamente e penetra facilmente nas células eucarióticas viáveis e 2) aqueles, tais como o MTS, XTT e WST-1, que são carregados negativamente e não penetram facilmente nas células e, por essa razão, são utilizados em conjunto com um aceitador de elétrons intermediário, tais como o metassulfato de fenazina (PMS), (BERRIDGE; HERST; TAN, 2005).

Figura 7 -

 7 Figura 7 -Estrutura química dos diferentes sais de tetrazólio e o aceitador de elétrons intermediário, metassulfato de fenazina (PMS).

Figura

  Figura 8 -Estrutura da SRB.

Figura 9 -

 9 Figura 9 -Vias de sinalização envolvidas na apoptose: via mitocondrial e via extrínseca.

  bioquímicas cuidadosamente coordenadas. Essa complexidade permite uma regulação precisa de todas as fases do ciclo celular e é essencial para garantir que a fase anterior seja completada sem erros (HOCHEGGER; TAKEDA;[START_REF] Hochegger | Cyclin-dependent kinases and cell-cycle transitions: does one fit all?[END_REF][START_REF] Khodjakov | The nature of cell-cycle checkpoints: facts and fallacies[END_REF][START_REF] Khodjakov | The nature of cell-cycle checkpoints: facts and fallacies[END_REF].Na maioria das células eucarióticas, três principais pontos de verificação estão envolvidos no controle da progressão do ciclo celular: (1) o ponto de verificação que ocorre no final da fase G1 é regulado principalmente por fatores de crescimento extracelulares e sinalizam se o ambiente é favorável para a proliferação celular antes de se comprometer com a fase S, (2) o ponto de verificação G2/M impede a entrada na mitose até que o DNA danificado seja reparado e a replicação do DNA seja concluída livre de erros, e (3) o ponto de verificação funciona durante a mitose entre a metáfase e anáfase e assegura a fixação adequada dos cromossomos replicados para os fusos mitóticos levando à conclusão da mitose e da citocinese[START_REF] Alberts | Biologia Molecular da Célula[END_REF][START_REF] Cooper | A Célula: uma abordagem molecular[END_REF][START_REF] Cooper | A Célula: uma abordagem molecular[END_REF][START_REF] Kastan | Cell-cycle checkpoints and cancer[END_REF][START_REF] Kastan | Cell-cycle checkpoints and cancer[END_REF].

  novas moléculas de ciclina (HOCHEGGER; TAKEDA;[START_REF] Hochegger | Cyclin-dependent kinases and cell-cycle transitions: does one fit all?[END_REF][START_REF] O'connor | Essentials of Cell Biology[END_REF][START_REF] O'connor | Essentials of Cell Biology[END_REF][START_REF] Schwartz | Targeting the cell cycle: a new approach to cancer therapy[END_REF][START_REF] Schwartz | Targeting the cell cycle: a new approach to cancer therapy[END_REF]. Quando os níveis de ciclinas diminuem, as CDKs correspondentes tornam-se inativas. A degradação das ciclinas é igualmente importante para que a progressão através do ciclo celular ocorra, e este processo ocorre pela ação de enzimas específicas que quebram as ciclinas em momentos definidos no ciclo celular (O'CONNOR; ADAMS, 2010).

  3. As M-ciclinas ativam Cdks que estimulam a entrada na mitose no ponto de verificação G2/M[START_REF] Alberts | Biologia Molecular da Célula[END_REF](ALBERTS et al., , p.1062)).As células eucarióticas contêm diferentes tipos e números de CDKs. Por exemplo, as leveduras tem apenas uma única CDK, enquanto que nos vertebrados quatro CDKs (CDK1, CDK2, CDK4 e CDK6) são consideradas como sendo as principais reguladoras do ciclo celular e contribuem para a indução e ou progressão das fases G1/S (CDKs 4 e 6), S (CDK2) e G2/M (CDK1) (DAVIDSON; NIEHRS, 2010; HOCHEGGER; TAKEDA; HUNT, 2008). Em reposta a estímulos mitóticos ou fatores de crescimento extracelulares, a expressão das ciclinas D (ciclinas D1, D2 e D3) é aumentada. Estas ciclinas associam-se as CDK4 e CDK6 e formam complexos ativos que promovem a entrada no ciclo celular em G1 (MALUMBRES; BARBACID, 2009). Posteriormente, a entrada na fase S é regulada pelo complexo ciclina E-CDK2 (HOCHEGGER; TAKEDA; HUNT, 2008; POEHLMANN; ROESSNER, 2010). Durante esta etapa ocorre um aumento da expressão da ciclina A, que em conjunto com CDK2 permite que a síntese de DNA ocorra. O complexo ciclina A-CDK2 é então responsável pela progressão através da fase S (PINTO; FELZENSZWALB, 2003; POEHLMANN; ROESSNER, 2010). A progressão de G2 para mitose requer a atividade de CDK 1. Durante o final da fase S e ao longo da fase G2, as células se preparam para entrar na mitose, aumentando desse modo, os níveis de ciclinas A e B (SCHWARTZ; SHAH, 2005). Com o aumento dos níveis das ciclinas A e B, formam-se os complexos ciclina A-Cdk1 e ciclina B-CdK1, que são responsáveis pela fosforilação de substratos, tais como o citoesqueleto, histonas e proteínas do envelope nuclear (KARP, 2010; SCHWARTZ; SHAH, 2005).

  Orbitide from the Latex of Croton urucurana Priscila de M. Cândido-Bacani, †, ‡ Patrícia de O. Figueiredo, † Maria de F. C. Matos, ‡ Fernanda R. Garcez, † and Walmir S. Garcez,* , † † Instituto de Química, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS 79074-460, Brazil ‡ Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS 79070-900, Brazil ABSTRACT: The bioactive ethyl acetate phase obtained from the latex of Croton urucurana Baillon afforded a novel orbitide (1), [19-NαC]-crourorb A1, that proved active against NCI-ADR/RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxorubicin (positive control) and inactive up to the highest concentration tested against nontumor NIH/3T3 cells. The structure elucidation was based on 1D and 2D NMR spectroscopy, further supported by HRESIMS data and by application of εarfey's method for determination of the absolute configuration of its amino acid residues. This is the first orbitide obtained from C. urucurana.

Figure 1 .

 1 Figure 1. [19-NαC]-crourorb A1 (1), the orbitide [(cyclo)-G a SL a G b AFG c NL b -] isolated from the latex of Croton urucurana.

( 2 , 4 - 1 .

 241 dinitro-5-fluorophenyl)-L-alaninamide (L-FDAA, εarfey's reagent, Sigma, 100 µL) was added and the mixture heated at 40 C for 1 h. The reaction mixture was cooled to RT, quenched by adding 2 N HCl (10 µL), dried, and dissolved in 50% aqueous CH 3 CN (600 µL). Five microliters of the FDAA derivative was subjected to HPLC using a C 18 column at RT(20 C). Aqueous CH 3 CN containing 4% TFA was used as the mobile phase, with linear gradient elution(20-35%, 40 min) for L-and Dserine retention time analysis and with isocratic elution (45% ACN) for L-and Dalanine, phenylalanine, and leucine retention time analysis at a flow rate of 1 mL min - FDAA-derived amino acids were detected by UV absorption at 340 nm.From each separate 50 mM aqueous solution of D-or L-configurations of A, S, F, or L, 50 µL aliquots were taken (totaling eight different samples) and 1 M NaHCO 3(20 µL) and a 1% solution of L-FDAA in acetone (100 µL) were added. The mixtures were separately heated at 40 C for 1 h, then cooled to RT, quenched by adding 2 N HCl (10 µL), dried, and dissolved in 50% aqueous CH 3 CN (600 µL). Subsequently, 5 µL of each FDAA derivative was analyzed by HPLC. The following retention times (min) were observed for the L-FDAA derivatives of the standard amino acids and for the residual εarfey's reagent, respectivelyμ δ-Ala(7.5 and 9.4), D-Ala(8.4 and 9.4),

Fig. 1S. 1 H

 1 Fig. 1S. 1 H NMR (400 MHz, DMSO-d 6 ) spectrum of compound 1.

Fig. 8S .

 8S Fig. 8S. Infrared spectrum of compound 1.

Fig. 10S .

 10S Fig. 10S. UV spectrum of compound 1.

  Cell proliferation Reagent WST-1(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate) was from Roche (Meylan, France) and SensoLyte Homogeneous AMC Caspase-γ/7 Assay' kit was from Anaspec (δe-Perray-en-Yvelines, France). Propidium Iodide (PI), Hoechst and Dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St-Quentin-Fallavier, France). Antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p70 S6 Kinase (Thr389), phospho-Akt (Ser473), Bad, Puma, Bid, Bax, Bak, Bim, cyclin B1, CDK1 and cyclin D1 were purchased from Cell Signaling Technology (Ozymz, Saint-Quentin-en-Yvelyne, France). Polyclonal antibodies against clived-caspase 3 and hsc-70 (sc-7298) were from Santa Cruz Biotechnology (Tebu-bio, Le-Perray-en-Yvelines, France). The MEK inhibitor U0126 was from Promega (Charbonnières, France), Wotmannin inhibitor was from Sigma-Aldrich (St-Quentin-Fallavier, France) and JNK inhibitor II was from Calbiochem (VWR, Fontenaysous-Bois, France). Secondary antibodies conjugated to horse-radish peroxidase were obtained from Dako (Trappes, France). Secondary antibodies coupled with Alexa Fluor were from Becton Dickinson (BD Biosciences, San Jose, CA). The Cisplatin was obtained from Mylan (Saint-Priest, France) and was used as a positive control. [19-NαC]-crourorb A1 was isolated and identified from Croton urucurana Baillon latex at Institute of Chemistry, Federal University of Mato Grosso do Sul (UFMS), CampoGrande, Brazil (Cândido-Bacani et al., 2015).

  A and B), while the percentage of cells in G 0 /G 1 phase was significantly decreased (p<0.01) (Figure 4 B).To better understand the mechanisms of crourorb A1-induced G2/M arrest, we examined G2/M and G1 phases related regulatory proteins by Western blot. As shown in Figure4C, the expression level of CDK1 (cyclin-dependent kinase 1), cyclin B and cyclin D1 increased in Huh-7 cells in a time-dependent manner. A slight decrease of CDK1 can be observed after 48h of treatment.JNK pathway, but not MEK/ERK and AKT, is involved in the mortality induced by Crourorb A1.The phosphorylation level of MAP kinases ERK1/2, JNK and p38 and proteins AKT and p-70 were analyzed in Huh-7 cells treated with crourorb A1 at 36µg/ml for 10min, 30min, 3h, 6h, 10h, 24h and 48h. As shown in Figure5A, crourorb A1 stimulated the phosphorylation of AKT after 3h of treatment with a slow increase thereafter until 48h. Early phosphorylations of ERK1/2 are obtained in control (DMSO) and crourorb A1 treated cells whereas ERK1/2 activation after 10h is specific to the crourorb A1 treated cells. This activation lasted until 48 h in a time dependent manner compared to the control. Interestingly, phosphorylation of JNK peaked at 30 min and decreased until 10 h (Figure5 A). Thereafter, no JNK phosphorylation could be detected. Phospho-70 and of phospho-38 were not greatly influenced by crourorb A1 during the time course analysed as compared to the control cells.

  spheroids, we performed 3D confocal imaging and Hematoxylin-Eosin staining of spheroids. Z-stack reconstructions of crourorb A1-treated spheroids in three dimensions (xyz) exhibited dramatic changes in cell morphology and in size of spheroids. After 48 h of treatment these morphological changes showed highly disorganized spheroids with more or less connected cells (Figure8A-B). These results were confirmed by Hematoxylin-Eosin staining (Figure8C-D). Changes of cell areas were measured in crourorb A1-treated cells after 8, 24, 32 and 48 h of treatment in 1.5mg/ml collagen gels. The results indicated that spheroids surface area decreased by 45% after 48h of treatment with crourorb A1 as compared to control DMSO-treated cells (Figure8 E).DiscussionCrourorb A1, a novel cyclic peptide identified from the medicinal Croton urucurana Baillon latex (Brazil), has previously been demonstrated as a candidate anticancer agent due its potential cytotoxic effects against a panel of human cancer cell lines[START_REF] Cândido-Bacani | Cytotoxic orbitide from the latex of Croton urucurana[END_REF]. However, the cellular and molecular mechanisms underlying responsible for crourorb A1-induced cytotoxicity on cancer cell lines have not been investigated. In the present study, we determined the cytotoxic effects of crourorb A1 on Huh-7 cells and we demonstrated that treatment of Huh-7 cells with crourorb A1 induced apoptosis, cell cycle arrest in G2/M and inhibited cell motility. We also demonstrated the effect of crourorb A1 on the viability Huh-7 cells grown in 3D collagen gels.The cytotoxicity of crourorb A1 in Huh-7 cells was detected by WST-1 assay, and results indicated that crourorb A1 decreased cell viability in a dose dependent and time-dependent manner. Crourorb A1 concentrations in the range of 50-200 g/ml reduced the cell viabilityν however no alteration in the viability was observed with lower crourorb A1 concentrations (0.γ5 and 1β.5 g/ml) after β4, 48 and 7β h of exposure. The IC 50 value of crourorb A1-treated Huh-7 cells at 48 hours (γ0.41 g/ml)

  have demonstrated that Astis, a family of cyclopentapeptides isolated from the roots of a medicinal plant, Aster tataricus (Compositae), exhibits potent caspases-dependent pro-apoptotic activity in human papillary thyroid carcinoma cells (NPA cell line). Recently, a new cyclic octapeptide, named cyclosaplin showed antiproliferative activity against human breast cancer cells (MDA-MB-231) by inhibition of cell viability and induction of apoptosis by caspase 3 activation (Mishra et al. 2014).

Figure 1 .

 1 Figure 1. Chemical structure of [19-NαC]-crourorb A1.

Figure 2 .

 2 Figure 2. Cell viability (%) in 2D cultures was determined by WST-1 assay. (A) Huh-7 cells were treated with increasing concentrations of crourorb A1 (0.35-200 µg/mL) or with 0.2% DMSO as control for 24, 48 and 72h. (B) Huh-7 cells were treated with increasing concentrations of crourorb A1 (10-70 µg/mL) or with 0.07% DMSO as control for 24h. Results are expressed as percentage change compared to the control ( ** p<0.01; significantly different from the control).

Figure 3 .

 3 Figure 3. Effects of crourorb A1 on apoptosis and necrosis in 2D Huh-7 cells. (A) Morphological changes of Huh-7 cells in the presence or absence of crourorb A1. Huh-7 cells were treated with 50 g/ml of crourorb A1 or with 0.05% DεSO as control

Figure 4 .

 4 Figure 4. Effects of crourorb A1 on cell cycle phase distribution in 2D Huh-7 cells. (A) Representative histograms depicting cell cycle distribution in Huh-7 cells treated with crourorb A1 (36 or 50 µg/mL) or with 0.05% DMSO as control for 24h and the cellcycle distribution was analyzed by flow cytometry. (B) The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle are shown. (C) For protein levels, cells were subjected to Western blot for analysis of the expression of CDK-1and cyclin B1

Figure 5 .

 5 Figure 5. Effects of crourorb A1 on cell signaling pathways in 2D Huh-7 cells. (A) Huh-7 cells were treated with 36 µg/mL of crourorb A1 or with 0.036% DMSO as control and arrested at the indicated times to analyze phosphorylated AKT, ERK1/2, JNK, p70S6 and p38 protein levels by Western Blot. Hsc-70 served as an internal control. (B and C) Cells were pretreated for 1 h with dimethyl sulfoxide or with the MEK inhibitor U0126 or wortmannin or JNK inhibitor II (and then exposed to 36µg/mL of peptide). (B) WST-1 assays were performed after 24 h of cotreatment with U0126 or wortmannin or JNK inhibitor II and crourorb A1. (C) AKT, ERK1/2 and JNK phosphorylations were monitored by western blot and Hsc-70 was used as an internal control. The data are represented as mean±SD of three independent experiments ( *** p<0.001).

Figure 6 .

 6 Figure 6. Crourorb A1 inhibits migration in 2D Huh-7 cells. Cells were cultured in the absence (A-B) or presence (C) of mitomycin C and treated with crourorb A1 (36 µg/mL) or with 0.036% DMSO as control for 24, 48 and 72h. The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch toward the center. Values are represented as mean±SD of three independent experiments ( * p<0.05; ** p<0.01, relative to those of the control group). Scale bar = 100µm.

Figure 7 .

 7 Figure 7. Cell viability (%) in 3D cultures (collagen gel 0.75 and 1.5 mg/mL) was determined by WST-1 assay. Huh-7 cells were treated with increasing concentrations of crourorb A1 (10-100 µg/mL) or with 0.1% DMSO as control for 24h after, 6 days of cultures. Results are expressed as percentage change compared to the control ( ** p<0.01; significantly different from the control).

Figure 8 .Figure 1 .

 81 Figure 8. Cell proliferation kinetics in 3D collagen gel cultures (collagen gel 1.5 mg/mL). (A) 3D reconstruction of Huh-7 cells treated with 0.062% DMSO as control or (B) 62µg/mL of crourorb A1 for 48h after, 4 days of cultures in 1.5 mg/mL collagen gel. For fluorescent dye labeling, cells were incubated with PBS containing 50 ε of RS19 for 20 minutes. (C) Hematoxylin and Eosin staining after paraffin inclusion and observation with slide scanner of Huh-7 spheroids treated for 48h with 0.062% DMSO as control or (D) 62µg/mL of crourorb A1 for 48h after, 4 days of cultures in 1.5 mg/mL collagen gel. (E) Huh-7 cells were treated with 62µg/mL of crourorb A1 or with 0.062% DMSO as control for 8h, 24h, 32h and 48h after, 4 days of cultures and cell surface (area) was measured ( ** p<0.01, *** p<0.001; significantly different from the control).

Figure 2 .

 2 Figure 2. Cell viability (%) in 3D cultures (collagen gel 0.75 and 1.5 mg/mL) was determined by WST-1 assay. Cisplatin (1-40 µg/mL) was employed as positive control for 24h after, 6 days of cultures. Results are expressed as percentage change compared to the control ( * p<0.05; ** p<0.01; significantly different from the control).

Figure 3 .

 3 Figure 3. Cisplatin (20 µg/mL) was employed as positive control by DEVD-AMC fluorometric assay ( *** p<0.001; significantly different from the control).

RÉSULTATSCHAPITRE 1 :CHAPITRE 2 : 7 )

 127 Croton urucurana Baillon (Euphorbiaceae), est une plante très connue et utilisée en médecine traditionnelle au Brésil, la «sangra-d'água» retrouvée dans l'État du Mato Grosso do Sul (région Centre-Ouest du Brésil). Son latex est abondamment utilisé en médecine traditionnelle dans le traitement de plusieurs types de cancer. En dépit de son importance ethnopharmacologique, on ne trouve aucune étude sur sa composition chimique ou sur sa potentielle activité anti-cancéreuse.δ'objectif de ce travail a été d'évaluer l'activité cytotoxique des extraits totaux, fractions d'extraits et de molécules purifiées, isolées du latex de C. urucurana. Nous avons étudié plus particulièrement les effets du crourorb A1 sur la viabilité, l'apoptose, le cycle cellulaire et la migration de cellules issues de carcinome hépatocellulaire humaine. Nous avons également évalué l'effet du crourorb A1sur la viabilité des cellules Huh-7 cultivées dans les gels de collagène en 3D. Le premier chapitre de cette thèse est consacré à l'isolement et à la détermination de la structure chimique d'un nouveau cyclopeptide isolé à partir du fractionnement chromatographique du latex de C. urucurana Baillon par des tests de cytotoxicité à la sulforhodamine B (SRB) sur six lignées cellulaires cancéreuses humaines. Cette nouvelle molécule a montré une activité cytotoxique importante sur ces cellules cancéreuses. Le second chapitre expose les résultats obtenus concernant les effets et l'évaluation du mécanisme d'action impliqué dans l'activité cytotoxique de [19-NαC]crourorb A1 sur les cellules de carcinome hépatocellulaire humain (Huh-7). Un nouvel orbitide cytotoxique extrait du latex de Croton urucurana Le latex de C. urucurana a été collecté dans l'habitat naturel de la plante durant le mois de Mars 2012 à Campo Grande, Brésil. La détermination botanique de cette espèce a été réalisée par le Pr. Arnildo Pott du Centre des Sciences Biologiques de la Santé de l'Universidade Federal de Mato Grosso do Sul, Brésil. L'étude phytochimique du latex de C. urucurana bioguidé et de son activité cytotoxique ont été réalisées par le test à la sulforhodamine B sur six lignées cellulaire cancéreuses : 786-0 (carcinome du rein), HT-29 (carcinome du colon), NCI-ADR/RES (adénocarcinome ovarien, phénotype de multirésistance), MCF-7 (adénocarcinome mammaire), PC-03 (carcinome de la prostate) et Hep-G2 (carcinome hépatocellulaire). δ'evaluation de l'effet cytotoxique de la molécule isolée à partir de latex á été également réalisée sur une lignée cellulaire non cancéreuse de fibroblastes murins, les cellules NIH/3T3. Les résultats de cytotoxicité ont été exprimés en concentration inhibitrice médiane inhibant 50% de la viabilité/croissance cellulaire (CI 50 ). δa fraction 'acétate d'éthyle' du latex a été fractionnée sur une colonne de Séphadex LH-20 et éluée par du MeOH en six fractions : de F1à F6. La fraction F2 est la plus active sur les lignées cellulaire 786-0 (CI 50 = 5,42 µg ml -1 ) et NCI-ADR/RES (CI 50 = 3,93 µg ml -1 ). Cette fraction a été analysée par HPLC semi préparative en mode isocratique qui a permis d'obtenir un composé majeur, le composé 1 dont l'identification et la structure moléculaire ont été déterminées par spectrométrie de masse. δe composé 1 a été obtenu sous forme d'un composé solide blanc amorphe. Son analyse par spectrométrie de masse haute résolution a montré la présence d'un ion pseudomoléculaire [M+H] + ayant un m/z 817,4199 correspond à un composé de formule moléculaire C 37 H 57 N 10 O 11 (calcul pour C 37 H 57 N 10 O 11 , m/z= 817,4203). Les spectres de résonance magnétique nucléaire (RMN) 1D 13 C et 1 H ont montré la présence de signaux caractéristiques d'un polypeptide. La détermination de la séquence d'acides aminés du composé 1 a été réalisée par utilisation de méthodes de spectrométrie de résonance magnétique nucléaire à deux dimensions ( 1 H et 13 C) et de spectrométrie de masse en tandem (MS/MS). δ'analyse des spectres RεN βD a permis d'identifier la présence de neuf résidus acides aminés: trois glycines (Ga, Gb et Gc), deux leucines (La, Lb), une phénylalanine (F), une asparagine (N), une alanine (A) et une sérine (S). Les corrélations observées dans les spectres COSY 1 H-1 H et NOESY ont permis de confirmer la nature cyclique de ce peptide et de déterminer la séquence des acides aminés, un cyclo -GaSLaGbAFGcNLb-. La caractérisation du composé 1 a été également confirmée par les données de spectrométrie de masse en tandem (MS/MS), montrant des fragments compatibles avec la séquence des résidus d'acides aminés identifiés. Ce composé 1, isolé pour la première fois du latex de C. urucurana, est un cyclononapeptide que nous avons nommé [19-NαC]-crourorb A1. Basé sur son squelette, il peut être classé comme un cyclopeptide de type Caryophyllaceae récemment redéfini comme un orbitide. δ'activité de [19-NαC]-crourorb A1 a été évaluée dans six lignées cellulaires cancéreuses humaines. Le composé présente une cytotoxicité puissante sur la lignée cellulaire cancéreuse NCI-ADR/RES (ovaire, le phénotype de multirésistance) (CI 50 = 4,8 µM) à un niveau équivalent à celle de la doxorubicine (témoin positif) (CI 50 = 5,1 µM), montrant que le composé 1 de la fraction 2 est en partie responsable de l'effet cytotoxique sur cette lignée cellulaire. Cependant, ce composé a une activité plus modérée sur les cellules 786-0 avec une CI 50 supérieure à la fraction 2. Ce composé ne peut donc porter en totalité l'activité cytotoxique observée pour le fraction 2 des cellules 786-0. Le composé 1 est de plus pratiquement inactif (CI 50 => 305,8 µM) sur la lignée non cancéreuse NIH/3T3, montrant une inhibition qui pourrait être sélective contre les cellules cancéreuses analysées. Nos résultats indiquent un potentiel thérapeutique anticancéreux du latex de C. urucurana. Cependant, des études complémentaires sont nécessaires pour déterminer les mécanismes d'action impliqués dans la cytotoxicité cellulaire du crourorb A1 sur des lignées de cellules cancéreuses humaines. Le [19-NαC]-crourorb A1 isolé du latex de Croton urucurana induit l'arrêt du cycle cellulaire et l'apoptose des cellules issues de carcinome hépatocellulaire humain Nous avons isolé et identifié un cyclononapeptide, nommé [19-NαC]-crourorb A1, à partir du latex de Croton urucurana. Dans ce travail, l'activité cytotoxique et les mécanismes d'action du crourorb A1 ont été analysés dans les cellules issues d'un carcinome hépatocellulaire humain, Huh-7. L'évaluation de la cytotoxicité in vitro du crourorb A1 (test wst1) sur les cellules Huh-7 a démontré que la viabilité des cellules est inhibée de manière dose et tempsdépendant : CI 50 de 35,75±1,00, 30,41±2,14 et 20,07±3,40 g/mL, respectivement après 24, 48 et 72h de traitement. δe Crourorb A1 induit des altérations morphologiques liées à l'apoptose après observation au microscope à fluorescence et marquage de l'ADN (Hoechst). Nous avons mesuré une forte activation des caspases 3 et 7 par le crourorb A1, sans progression nécrotique. Afin de déterminer les mécanismes possibles responsables de la mort cellulaire apoptotique induite par le crourorb A1, l'expression des protéines pro-apoptotiques de la famille Bcl-2 a été analysée par Western blot. Nos résultats indiquent que le crourorb A1 provoque une nette augmentation de l'expression des protéines pro-apoptotiques (Bak, Bid, Bax, Puma, Bim et Bad) ainsi que de la caspase 3 clivée. L'ensemble de ces résultats démontrent que le crourorb A1 exerce un effet cytotoxique en induisant une apoptose caspase-dépendante. Nous avons également déterminé que la voie de signalisation JNK-MAPkinase semble nécessaire à l'induction de la mort cellulaire des cellules Huh-7 traitées au crourorb A1. δ'analyse de la distribution des cellules Huh-7 dans les différentes phases du cycle cellulaire (marquage à l'iodure de propidium et analyse par cytomètrie de flux) montre que le Crourorb A1 induit un arrêt du cycle cellulaire en phase G 2 /M. Simultanément, nous avons observé une diminution du nombre des cellules Huh-7 en phase G 0 /G 1 . En parallèle, les expressions de la cycline-dépendante kinase CDK1, des cyclines B1 et D1 sont augmentées après traitement au crourorb A1. De plus, le Crourorb A1 a un effet inhibiteur significatif de la migration des cellules Huh-7, en présence ou absence de mitomycine C.Nous avons également confirmé, l'effet inhibiteur du crourob A1 sur la viabilité des cellules Huh7 cultivées en 3D dans les gels de collagène 1 et démontré que les cellules cultivées en 3D semblent plus résistantes au crourorb A1 par rapport aux cultures en monocouche 2D. Des IC 50 de 62,10 ± 4,70 µg/mL et 62,52 ± 3,12 µg/mL (après 24h de traitement) ont été obtenues respectivement avec les cellules cultivées dans des gels de collagène à 0,75 mg/mL (1Pa) et 1,5 mg/mL (3.5Pa) démontant une plus forte résistance des cellules Huh7 au crourob A1 en culture 3D par rapport aux cultures 2D.A partir dos resultados obtidos no estudo fitoquímico guiado pela atividade citotóxica frente a linhagens de células neoplásicas humanas, podemos concluir que: 1) O extrato etanólico das cascas do caule de Croton urucurana Baillon foi inativo até a maior concentração testada (GI 50 = >250 µg/mL) em todas as linhagens de células neoplásicas humanas; 2) O látex de C. urucurana foi ativo em todas as linhagens de células neoplásicas humanas testadas e foi selecionado para a continuidade ao estudo fitoquímico biomonitorado; 3) O estudo fitoquímico biomonitorado da fase acetato de etila do látex por meio da atividade citotóxica frente a linhagens de células neoplásicas humanas levou ao isolamento de um composto majoritário, o ciclopeptídeo [19-NαC]-crourorb A1, inédito na literatura; 4) [1-9-NαC]-crourorb A1 se mostrou potente para a linhagem celular de adenocarcinoma de ovário resistente (NCI/ADR-RES) com um valor de GI 50 =3,98 µg/mL e foi tão potente quanto a doxorrubicina (controle positivo), e inativo para a linhagem de células não neoplásicas de fibroblasto murino (NIH/3T3) até a maior concentração testada (GI 50 = >250 µg/mL) sendo seletivo para a linhagem NCI/ADR-RES; A partir dos resultados obtidos nos diferentes sistemas-teste para elucidar os mecanismos responsáveis pela citotoxicidade induzida por [19-NαC]-crourorb A1 em células de hepatocarcinoma celular humano (Huh-7), podemos concluir que: 1) A citotoxicidade de [19-NαC]-crourorb A1 em células Huh-7 se mostrou dose e tempo dependentes; 2) [19-NαC]-crourorb A1 induziu apoptose em células Huh-7 e estes efeitos foram atribuídos ao aumento da atividade de caspases 3/7 e aumento da expressão de proteínas próapoptóticas. A presença de células com alterações morfológicas características da apoptose pôde ser visualizada quando observadas por microscopia de fluorescência e não foi observado nenhum efeito na necrose em células Huh-7; 4) [19-NαC]-crourorb A1 induziu o acúmulo de células na fase G2/M do ciclo celular e interferiu na expressão de proteínas que participam do ciclo celular, aumentando o nível da expressão da cinase dependente de ciclina (CDK1), Ciclina B1 e Ciclina D1. 5) [19-NαC]-crourorb A1 inibiu a migração de células Huh-7 tempo dependente. 6) [19-NαC]-crourorb A1 estimulou a fosforilação da ERK1/2, JNK e AKT de maneira tempo-dependente e não influenciou nos níveis de fosforilação da p38 e p70S. A via JNK está envolvida no mecanismo de morte celular induzida por [19-NαC]-crourorb A1 em células Huh-7. As células cultivadas em geis de colágeno em 3D apresentaram maior resistência ao [19-NαC]-crourorb A1 quando comparada com a cultura de células em 2D; no entanto não foi observada diferença para ambos os modelos de cultura em 3D.Tabela 1 -Obtenção das fases Hexânica, Acetato de Etila e Hidrometanólica a partir de 55 g do látex de Croton urucurana Baillon. foi mantida em solução para evitar a degradação de seus constituintes químicos (taninos).

Figura 1 -

 1 Figura 1 -Fracionamento biomonitorado do látex de C. urucurana por meio do ensaio de citotoxicidade com o corante sulforrodamina B.

Figura 2 -

 2 Figura 2 -Perfil cromatográfico da Fração 2 por CLAE. O pico 3 corresponde a substância 1 isolada.

  

  

  

  

  

  

  

  2.1 Plantas medicinais como fonte de agentes anticâncer ........................................ 2.2 Plantas medicinais e o gênero Croton ................................................................. 2.2.1 Croton urucurana Baillon .................................................................................. 2.2.1.1 Características botânicas, farmacológicas e composição química ................

2.3 Cultura de células ................................................................................................ 2.4 Ensaios de citotoxicidade in vitro ........................................................................ 2.5 Morte celular -apoptose e necrose ..................................................................... 2.5.1 Via intrínseca .................................................................................................... 2.5.2 Via extrínseca ................................................................................................... 2.6 Ciclo celular .........................................................................................................

1 Plantas medicinais como fonte de agentes anticâncer

  

	Baillon (Euphorbiaceae) conhecida popularmente como "sangra d´água" que tem A descoberta de medicamentos oriundos de plantas medicinais tem
	sido utilizado na medicina popular sul-mato-grossense no tratamento de vários tipos desempenhado um papel importante no tratamento, cura e prevenção de um amplo
	de câncer e, apesar de sua importância etnofarmacológica, não há relatos de espectro de doenças, especialmente do câncer (BUTLER, 2004; CRAGG;
	estudos relacionados à sua composição química ou atividades anticâncer na GROTHAUS; NEWMAN, 2014; CRAGG; NEWMAN, 2013; ROCHA; LOPES;
	literatura. SCHWARTSMANN, 2001). Entre as substâncias utilizadas no tratamento do câncer,
	Reconhecendo a importância da espécie C. urucurana no tratamento do 60% são derivadas direta ou indiretamente de produtos naturais (NEWMAN;
	câncer pela população do estado e tendo em vista a ausência de critérios científicos CRAGG; SNADER, 2003).
	Vincristina; R= CHO Vimblastina; R=CH3	Taxol
	Etoposídeo	Teniposídeo

Associada ao conhecimento popular, uma metodologia utilizada na busca de substâncias com potencial atividade anticâncer é o estudo biomonitorado ou screening, o qual visa ao isolamento de compostos de interesse farmacológico a partir do fracionamento biomonitorado de um extrato que apresentar resultados positivos no teste de citotoxicidade utilizando linhagens de células tumorais humanas (CRAGG; GROTHAUS; NEWMAN, 2009; YOUNES; VARELLA; SUFFREDINI, 2007). Essas abordagens podem ser vistas como complementares e a sua utilização no Brasil, especialmente no estado de Mato Grosso do Sul, se torna uma ferramenta importante, visto que o mesmo possui uma riqueza inestimável, representada pela biodiversidade de espécies vegetais nos Biomas Cerrado e Pantanal, muitas de uso etnofarmacológico. Dentre essas espécies, destaca-se o látex de Croton urucurana para o uso desta planta, decidiu-se inicialmente realizar o estudo fitoquímico biomonitorado por meio da atividade citotóxica frente a linhagens de células tumorais humanas, visando ao isolamento e a identificação das substâncias com potencial atividade anticâncer. Estas etapas foram realizadas no Laboratório de Pesquisa de Produtos Naturais Bioativos (ProNaBio) e no Laboratório de Biologia Molecular e Culturas Celulares da Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS. Adicionalmente, foram estudados os mecanismos celulares e moleculares implicados na atividade citotóxica da substância isolada em uma linhagem de hepatocarcinoma celular humano. Esta etapa foi realizada durante o estágio de doutoramento em Rennes (França) no Institut de Recherche Santé, Environnement & Travail (IRSET), da Université de Rennes 1, sob a orientação do Prof. Dr. Georges Baffet, responsável pela equipe Signalisation et Modélisation. 2.Drogas anticâncer de origem vegetal em amplo uso clínico incluem os alcaloides da vinca isolados de Catharanthus roseus (L.) G. Don (vincristina e vimblastina); etoposídeo e teniposídeo, que são derivados semissintéticos da epipodofilotoxina (um isômero da podofilotoxina) isolada de Podophyllum peltatum Linnaeus e Podophyllum emodi Wallich; o taxol derivado de Taxus brevifolia Nuttall ("teixo") e os derivados da camptotecina, um alcaloide extraído de uma árvore ornamental chinesa, Camptotheca acuminata Decaisne. Todos estes compostos são citotóxicos e agem principalmente inibindo a proliferação celular através de mecanismos celulares distintos (CRAGG; NEWMAN, 2005; CRAGG; NEWMAN, 2013; ROCHA; LOPES; SCHWARTSMANN, 2001) (Figura 1).

Figura 1 -Estruturas de algumas drogas anticâncer derivadas de plantas em uso clínico. Fonte: Cragg; Newman (2005).

Espécie Uso tradicional Atividades farmacológicas de diferentes partes da planta e/ou compostos relevantes isolados

  

	e alcaloides (GUPTA; BLEAKLEY; GUPTA, 2008; SALATINO; SALATINO; NEGRI, 2007). Muitas espécies de Croton produzem óleos essenciais ricos em mono e Figura 2 -Espécies de Croton, usos tradicionais e efeitos farmacológicos de partes das plantas e alguns compostos importantes isolados.
	sesquiterpenoides, como também fenilpropanoides (PALMEIRA JÚNIOR et al.,
	2006). Além destes metabólitos, foram isolados e caracterizados ciclopeptídeos das
	espécies Croton gossypifolius (QUINTYNE-WALCOTT; MAXWELL; REYNOLDS, 2007) e Croton sparsiflorus (MEHMOOD; MALIK, 2010). O gênero é promissor em pesquisas de compostos naturais com atividades biológicas, tanto devido à diversidade de constituintes químicos, quanto pela importância farmacológica atribuída as suas espécies. Estudos realizados com espécies pertencentes a este gênero, também utilizadas na medicina popular, têm revelado resultados promissores quanto a suas atividades farmacológias (SALATINO; SALATINO; NEGRI, 2007) (Figura 2). Croton arboreous Anti-inflamatório Quatro sesquiterpenos → anti-inflamatória C. cajucara Diabetes, hipercolesterolemia, distúrbios digestivos, alterações hepáticas, perda de peso Óleo essencial da casca → cicatrização da úlcera gástrica, antileishmanioseν extrato aquoso da casca → redução do ganho de peso e maior sensibilidade dos adipócitos para isoprenalina e adrenalina; trans-crotonina, trans-desidrocrotoninaν ácido acetilaleuritólico → efeitos hipolipemidemico e hipoglicemicoν trans-desidrocrotonina → anti-estrogênio, anticâncer; linalol → antibacteriana e antifúngica C. celtidifolius Inflamação, leucemia, úlceras, reumatismo casca → anti-inflamatória e antioxidante C. eluteria Bronquite, febre, malária, digestivo, Extratos da casca → estimulação da secreção gástrica hipertensão C. kongensis Dismenorreia Diterpenos secocauranos → citotóxica, anti-micobacteriana e anti-malarial C. lechleri Hemostático, cicatrização de feridas, purgativo δátex vermelho → anti-inflamatória, antiviral, antibacteriana, antileucemia; SP-γ0γ → anti-viral: vírus sincicial respiratório, herpes simplex, genital e anogenital C. macrostachys Purgativo e diabetes Sementes e raízes → purgativa C. malambo Dor, reumatismo, inflamações, diarreia, Extrato da casca → antinociceptiva e anti-inflamatória diabetes, úlcera gástrica C. nepetaefoilius Estomacal, flatulência, cólica intestinal, Óleo essencial → antiespasmódicaν cineol, metileugenol → miorrelaxante e antiespasmódica aperitivo C. oblongifolius Muitas aplicações, incluindo alargamento do Extrato → atividade antihepatotóxicaν diterpenos → citotóxica fígado, febre e platelmintos C. palanostigma Inflamação intestinal, cicatrização de feridas, Alcaloide taspina → citotóxica úlceras gástricas C. schiedeanus Hipertensão Extrato aquoso e etanólico; quercetina-3,7-dimetil éter → vasorelaxante e anti-hipertensiva C. tonkinensis Dor de estômago, dispepsia, úlceras gástrica Diterpenos → citotóxica e duodenal, lepra, psoríase, urticária C. sublyratus Anti-helmínticos e problemas dermatológicos Plaunotol → atividade antiúlcera, anticâncer, antibacteriana
	Fonte: Adaptado de Salatino; Salatino; Negri (2007). C. urucurana Analgésico, inflamação, infecção cicatrização de feridas, câncer C. zambesicus Febre, malária, convulsões, hipertensão, infecções microbianas, disenteria C. zehntneri Distúrbio nervoso, anorexia, distúrbio gastrointestinal, edulcorantes	Casca e látex vermelho → antidiarreica, antifúngicaν catequina, ácido acetilaleuritólico → antibacterianoν ácido acetilaleuritólico → analgésica Folhas → citotóxicaν diterpeno → citotóxica Óleo essencial → relaxante muscular intestinal, efeito depressor central, antinociceptivaν anetol e estragol → relaxante muscular intestinal; anetol → anticâncer
	Entre as partes da planta utilizadas para fins medicinais encontram-se as folhas,
	cascas do caule e o látex (GUPTA; BLEAKLEY; GUPTA, 2008; SALATINO;
	SALATINO; NEGRI, 2007).	
	Como constituintes químicos isolados das espécies de Croton é citado uma
	enorme diversidade de compostos biologicamente ativos como terpenos, flavonoides

2.2.1 Croton urucurana Baillon 2.2.1.1 Características botânicas, farmacológicas e composição química

  

	Croton urucurana Baillon é uma espécie arbórea nativa, pioneira, decídua,
	heliófita, encontrada frequentemente em matas ciliares de várias formações
	florestais (LORENZI, 2008). É característica de terrenos muito úmidos e pantanosos
	(LORENZI; MATOS, 2008), ocorrendo também em formações secundárias, como
	capoeiras e capoeirões (LORENZI, 2008). Esta espécie é endêmica em regiões
	tropicais e subtropicais da América do Sul com ampla dispersão pelo Brasil, onde
	ocorre nos estados da Bahia, Rio de Janeiro, Minas Gerais, Mato Grosso do Sul,
	Paraná, Santa Catarina e Rio Grande do Sul (CORDEIRO

  Table 1 depicts the cytotoxicity results obtained for 1 and the foregoing extracts, phases, and fractions. HRESIMS of 1 showed an [M+H] + ion at m/z 817.4199, compatible with the molecular formula C 37 H 57 N 10 O 11 (calcd for C 37 H 57 N 10 O 11 , m/z 817.4203). 13 C NMR data revealed four signals for a benzene ring [ C 126.6, 128.3 (×2), 129.4 (×2), 137.2], in addition to signals between C 60.1 and 15.9 and 10 signals between C 174.4 and 169.2. These 10 signals, attributable to amide carbonyl resonances, suggested that 1 is a peptide. The 1 H NMR spectrum was consistent with this possibility, based on resonances between H 5.05 and 0.82 and on a group of signals between H 8.84 and 7.00, characteristic of hydrogens linked to amide nitrogens (or peptide linkages) and of aromatic hydrogens (Table

Table 1 . In Vitro Cytotoxicity (GI 50 ) of Latex Extracts, Phases, Fractions, and Compound 1 from Croton urucurana against Six Human Cancer Cell Lines. a

 1 

	a GI 50 : concentration that inhibits cell growth by 50%. Cancer cell lines: 786-0 (kidney), HT-29 (colon), MCF-7 (breast), NCI-ADR/RES (ovary, multidrug-resistance phenotype), Hep-G2 (hepatocellular), and PC-03 (prostate). NT: not tested. Doxorubicin was the positive control. Values represent means obtained
	from three independent experiments, each run in triplicate.

Table 2 . 1D and 2D NMR Data for [19-NαC]-Crourorb A1 (1) in DMSO-d 6 . a

 2 

	C/H Glycine A (G a ) NH α α CO Serine (S) NH OH ,  α CO	C 42.81 169.19 60.12 58.11 174.41	H 8.57, br s 3.76 overlapped 3.59 dd, J 17.0, 4.6 Hz 8.84, br s 5.05, t J 6.0 Hz 3.69 (2H), m 3.88, t J 6.0 Hz	HMBC H-C 169.19 G a CO 174.71 SCO 174.71 SCO	NOE 8.84 SNH, 3.88 Sα, 3.59 G a α, 3.76 G a α, 7.22 LB2 8.57 G a NH, 7.22 LB NH 7.22 LBNH 4.55 L a α, 8.57 G a NH, 3.88 Sα, 7.γγ L a NH 8.84 SNH	COSY 3.59 G a α, 1 H-1 H 3.76 G a α 8.57 G a NH, 3.59 G a α 8.57 G a NH, 3.76 G a α 3.88 Sα 3.69 S , S  5.05 SOH, 8.84 SNH
	Leucine A (L a )					
	NH		7.33, br s		NH 1.75 L 7.97 G b NH, 8.84 S	4.55 L a α
	  	23.16 21.39 24.10	0.90, d, J 6.5 Hz 0.82, d, J 6.5 Hz 1.53, m 1.75, br t J 12.0 Hz	21.39 L a , 39.80 L a 23.16 L a , 39.80 L a 170.97 L a CO, 50.35 L a α	0.82 L a , 1.53 L a 0.90 L a , 4.55 L a α 0.90 L a 7.33 L a NH, 1.45 L a , 4.55 L a α	1.53 L a 1.53 L a 0.90 L a , 0.82 L a  4.55 L a α, 1.45 L a 
	 α CO Glycine B (G b ) NH α α CO Alanine (A) NH	39.80 50.35 170.97 43.08 169.22	1.45, br dd J 12.0, 8.4 Hz 4.55 ddd J 11.4, 8.4, 2.8 Hz 7.97, t J 5.9 Hz 3.82, overlapped 3.43, dd J 17.5, 5.9 Hz 8.52, br s	170.97 L a CO, 50.35 L a α 170.97 L a CO 169.22 G b CO 169.22 G b CO	7.33 L a NH, 1.75 L a a, 4.55 L a α 1.45 L a , 1.75 L a , 0,82 L a , 7.33 L a NH, 8.84 SNH 3.82 G b α, γ.4γ G b α, 7.33 L a NH, 8.52 ANH, 1.22 A 7.97 G b NH,7.33 L a NH 7.97 G b NH, 7.33 L a NH 4.24 Fα, γ.80 Aα,	4.55 L a α, 1.75 L a 7.33 L a NH, 1.75 L a , 1.45 L a  3.82 G b α, 3.43 G b α 7.97 G b NH, 3.43 G b α 7.97 G b NH, 3.82 G b α 3.80 Aα

a , 1.45 L a , 1.53 L a , 4.55 L a α, 3.82 G b α, γ.4γ G b α, a Recorded at 300.13/75.47 MHz. Chemical shifts referenced to residual DMSO.

Table 3 . Main Fragment Ions Observed in the HRESIMS/MS Spectrum of 1 (m/z 817.4199, [M+H] + , 46.3 eV).

 3 

	m/z	Fragment

of n amino acid residues. This ion can lose carbon monoxide to form an 'a' ion. The symbol J is for the N-terminal amino acid residue and Z designates the C-terminal amino acid residue, both of which result from bond cleavage. +

  The present results indicate therapeutic anticancer potential for Croton urucurana latex, corroborating its popular use in anticancer therapy. However, further studies are necessary to elucidate the mechanisms of action underlying the cytotoxic effects of compound 1 against human cancer cell lines.(27.2 mg) was obtained as the major component of the bioactive F2 fraction (100.8 mg) after isocratic semi-preparative HPLC using an RP-C 18 column (MeCN-H 2 O, 37:63, v/v), at a flow rate of 14 mL min -1 .

	[19-NαC]-Crourorb A1 (1): white, amorphous solid (H 2 O-MeCN); [α] 23 D = -
	163 (c 3 × 10 -2 , H 2 O-εeCN γ7%)ν UV (εeOH) max (log ) β0γ (γ.γβ), β45 (β.71) nm.
	EXPERIMENTAL SECTION IR (KBr) max 3300, 3051, 2950, 1652, 1548; 1 H and 13 C NMR data, see Table 2;
	General Experimental Procedures positive HRESIMS m/z 817.4199 [M+H] + (calcd for C 37 H 57 N 10 O 11 , m/z 817.4203);
	Optical rotation was determined on a PerkinElmer 341 polarimeter (Na filter, = 589
	nm). UV spectra were obtained on a USB4000 UV/vis spectrophotometer (Ocean
	Optics). IR spectra were recorded on a Nicolet iS5 FT-IR spectrometer (Thermo
	Scientific). NMR spectra were obtained in DMSO-d 6 (Cambridge Isotope
	Laboratories) on a Bruker DPX-300 spectrometer operating at 300.13 MHz
	( 1 H)/75.47 MHz ( Plant Material
	Bark and red latex of Croton urucurana Baillon (Euphorbiaceae) was collected from
	Campo Grande, Mato Grosso do Sul, Brazil (20β5 1γ S/ 5439 γ1 W) in εarch
	2012. The specimen was identified by Prof. Arnildo Pott (Centro de Ciências
	Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo
	Grande, MS, Brazil) and a voucher specimen (no. 30924) deposited at the CGMS
	Herbarium of the Universidade Federal de Mato Grosso do Sul.
	Cyclopeptide Extraction and Isolation
	Bark material (2500 g) was powdered and extracted in 95% EtOH at room
	temperature (RT) and subsequently concentrated under reduced pressure. The latex
	(55 g) was suspended in MeOH-H 2 O (9:1), and the mixture extracted successively

13 C)

. HRESIMS data were acquired in the positive ion mode on a micrOTOF-Q II instrument (Bruker Daltonics). Sephadex LH-20 was used for column chromatography. Reversed-phase semipreparative HPLC separation was performed with a Shimadzu LC-6AD pump using RP-18 silica gel (5 m, 21.6 × 250 mm) in a Shim-Pack column at a flow rate of 14 mL min -1 , with monitoring at 270 nm.

with hexane and EtOAc. Of the resultant fractions, the bioactive EtOAc phase was selected for further bioactivity-guided fractionation. A 3.0 g aliquot of this phase was introduced into a Sephadex LH-20 chromatography column

(5 × 36 cm, MeOH)

, to give six fractions (F1-F6).

Compound 1

Table 1 .

 1 IC 50 values, given in µg/mL, for crourorb A1 on 2D Huh7 cell lines.

			IC 50 (µg/mL) * ±SD	
		24h	48h	72h
	Crourorb A1	35.75±1.00	30.41±2.14	20.07±3.40
	Cisplatin	23.19±0.21	4.26±1.44	2.26±0.24

*

IC 50 values were expressed as the concentration that inhibited the growth of cells by 50%. Results are expressed as the mean of three independent experiments ± SD. Cisplatin was employed as positive control.

Table 2 .

 2 IC 50 values, given in µg/mL, for crourorb A1 on 3D Huh-7 cell lines.

	Days of cultures Collagen gel (mg/mL)	IC 50 (µg/mL)±SD *
			Crourorb A1	Cisplatin
	6	0.75 1.5	62.10±4.70 62.52±3.12	42.50±6.30 36.48±0.77

*

IC 50 values were expressed as the concentration that inhibited the growth of cells by 50%. Results are expressed as the mean of three independent experiments ± SD. Cisplatin was employed as positive control.

  Tabela 2 -Frações obtidas a partir de 3 g da Fase Acetato de Etila do látex de C. urucurana.

	Frações	Massa (mg)	Rendimento (%)
	F1	55,0	1,83
	F2	143,5	4,78
	F3	187,1	6,24
	F4	334,7	11,16
	F5	171,7	5,72
	F6	291,1	9,70

REVISÃO DE LITERATURA

OBJETIVOS

ACKNOWLEDGMENTS

This work was supported by Fundect-MS PRONEM 0088/12, CNPq, CPq-PROPP-UFMS and CAPES for the doctoral grants awarded to P.M. C.-B. Thanks are also extended to Prof. M. G. Carvalho, who kindly provided us with εarfey's reagent and the D-and L-amino acid standards; Prof. J. E. Carvalho and Prof. I. M. S Cólus, for providing the cell lines; and Prof. A. Pott, for his assistance in plant identification.

Acknowledgements

The authors wish to thank Fundect-MS, CNPq, and CPq-PROPP-UFMS for their financial support and CAPES for the doctoral grants awarded to P.M. Cândido-Bacani (Proc. nº 99999.002264/2014-05). Thanks are also extended to Dr M. Blanchard-Desce (UMR CNRS 5255, University of Bordeaux, France) who kindly provided us with fluorescent dye RS19 and platforms PIXEL, Flow cytometry and H 2 P 2 (SFR Biosit, UMS CNRS 3480 /US INSERM 018, Rennes).

Mato Grosso do Sul (FUNDECT), ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e a Pró-reitoria de Pesquisa e Pós-Graduação (CPq-

CAPÍTULO I:

CYTOTOXIC ORBITIDE FROM THE LATEX OF CROTON URUCURANA

Artigo publicado na Journal of Natural Products, 78: 2 -2 0, 2015 DOI:10.1021/acs.jnatprod.5b00724

AUTHOR INFORMATION

Corresponding Author

*Tel (W. S. Garcez): walmir.garcez@ufms.br.

Notes

The authors declare no competing financial interest. Key-words: Peptide, Croton urucurana, Cell cycle arrest, Apoptosis and Huh-7 cells Huh-7 spheroids appear as acini-like structures with hollow lumen reducing the drug accessibility hypothesis [START_REF] Bomo | Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment[END_REF].

SUPPORTING INFORMATION

CAPÍTULO II:

In conclusion, our data suggest that exposure of Huh-7 cells to crourorb A1, a cyclic peptide, resulted in decreased cell viability mediated by G2/M cell cycle arrest and consequent induction of apoptosis. Furthermore, crourorb A1 inhibited the cell migration. We also demonstrated that the JNK pathway is involved in the mechanism of crourorb A1-induced cell death in Huh-7 cells.

Conflicts of interest

The authors declare that there are no conflicts of interest.

CAPÍTULO III:

UN 

CONCLUSÕES

REFERÊNCIAS APÊNDICES