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UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
INSTITUTO DE CIÊNCIAS BÁSICAS DA SAÚDE
DEPARTAMENTO DE BIOQUÍMICA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS:
BIOQUÍMICA
A esponja marinha Polymastia janeirensis como
fonte de novos fármacos contra o câncer
Doutorando: Mário Luiz Conte da Frota Junior
Orientadores: Dr. José Cláudio Fonseca Moreira
Dra. Amélia Teresinha Henriques (co-orientadora)
Tese submetida ao Programa de Pós-
Graduação em Ciência Biológicas: Bioquímica,
como requisito para obtenção do grau de
Doutor em Bioquímica
Porto Alegre, 2008
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“ Imagination is more important than knowledge.
Knowledge is limited,
imagination encircles the world.”
(Albert Einstein)
I
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AGRADECIMENTOS
A minha esposa, Ana Lúcia Coelho Marques da Frota, pelo amor incondicional e
pelo apoio em todas as minhas decisões;
Aos meus pais, que me ensinaram os princípios morais e éticos para que eu
chegasse até aqui;
Ao meu irmão e melhor amigo, Michael Frota, minha fonte inspiradora;
A família Marques, pelo carinho e pela credibilidade;
A todos os colegas do Centro de Estudos em Estresse Oxidativo (Laboratório 32,
Departamento de Bioquímica, UFRGS). Horas de chimarrão, infinitas discussões,
inúmeras diferenças, incontáveis gargalhadas; Não conheço uma palavra que expresse o
que vocês são para mim;
Aos colegas do Laboratório 22, Departamento de Bioquímica, UFRGS; aos
colegas do laboratório de farmacognosia, Faculdade de Farmácia, UFRGS; aos colegas
da Fundação Zoobotânica do Rio Grande do Sul, Museu de Ciências Naturais;
Ao PPG-Bioquímica (UFRGS), às agências financiadoras - CAPES, CNPq,
PROPESQ/UFRGS e FAPERGS - bem como aos funcionários do Departamento de
Bioquímica da UFRGS;
II
Aos professores do PPG-Bioquímica da UFRGS;
Aos pesquisadores Dr. Andrés Delagado-Cañedo, Dra. Miriam Apel e Dra. Ana
Battastini, pela qualificada orientação e pelas oportunidades;
As pesquisadoras Cléa Lerner e Beatriz Mothes, pelo acolhimento desde o
nascimento desse trabalho;
Um agradecimento especial ao Dr. Fábio Klamt, que me ensinou muito do que
eu sei fazer hoje no laboratório, e que é um exemplo a ser seguido;
A minha co-orientadora, Dra. Amélia Teresinha Henriques, pela ajuda
incondicional no desenvolvimento dessa Tese;
E por último, mas não menos importante, a uma pessoa que consegue guiar um
bando de malucos para os melhores caminhos da vida. Um amigo, um irmão, um pai.
Uma vez ele disse que o que fazemos está longe de ser ciência, no sentido nobre da
palavra: “... fazemos pesquisa, e de qualidade!”. Dr. José Cláudio Fonseca Moreira, o
Zé, me ensinou a fazer pesquisa no sentido pleno da palavra. Ensinou-me quase tudo
nesse laboratório; senão tudo. Caráter, ética, profissionalismo, admitir o erro quando
estivermos errados, assumir a culpa, pedir desculpas, chamar a “responsa”. Se existe
alguém que está muito mais perto do que qualquer outra pessoa que eu conheço nesse
meio científico (cheio de “egos e de dedos”) de fazer ciência és tu Zé, e isso é uma
honra para quem tem a oportunidade de fazer parte do teu grupo de pesquisa. Muito
obrigado!
III
ÍNDICE
Resumo….....................……………............................................................……………1
Abstract............................................................................................................................2
Lista de abreviaturas.......................................................................................................3
Lista de Figuras...............................................................................................................4
Apresentação....................................................................................................................5
I. Introdução.....................................................................................................................6
II. Objetivos....................................................................................................................17
III. Artigos Científicos
III. 1 Artigo 1: Brazilian marine sponge Polymastia janeirensis induces apoptotic cell
death in human U138MG glioma cell line, but not in a normal cell culture…….……..20
III. 2 Artigo 2: Brazilian marine sponge Polymastia janeirensis induces oxidative cell
death through a caspase-9-apoptotic pathway in human U138MG glioma cell line…...29
III. 3 Artigo 3: Anti-proliferative activities of extracts and isolated compounds from
Polymastia janeirensis…………………...……………………………………………..54
IV. Discussão..................................................................................................................65
V. Conclusão...................................................................................................................74
VI. Perspectivas.............................................................................................................76
Referências Bibliográficas............................................................................................78
Anexos.............................................................................................................................85
IV
RESUMO
Nos últimos anos, o ambiente marinho tem sido pesquisado para uma variedade
de compostos com diferentes atividades biológicas. Entre todos os organismos
marinhos, as esponjas representam uma das fontes mais promissoras de novas drogas
contra o câncer. Embora o litoral brasileiro seja o segundo mais extenso depois da
Austrália, existem poucos estudos com esponjas marinhas coletadas no Brasil
objetivando a descoberta de novos fármacos. Nesse trabalho, nós investigamos os
efeitos de diferentes extratos da esponja marinha Polymastia janeirensis na viabilidade
de uma linhagem de glioma humano (U138MG). Além disso, avaliamos a seletividade
do efeito, caracterizamos os mecanismos envolvidos no processo de morte celular e
isolamos as frações ativas responsáveis pelas atividades encontradas. De uma maneira
geral, um efeito antiproliferativo foi observado quando as células forma expostas aos
extratos (aquoso e orgânico) da esponja. Entretanto, as doses mais altas (50 e 100
µg/ml) foram extremamente citotóxicas, inibindo mais de 80% a proliferação e a
viabilidade celular. Além disso, uma morte celular por necrose foi observada com essas
doses, enquanto uma morte celular por apoptose foi observada com a dose mais baixa
(10 µg/ml). Nós também demonstramos que a rota apoptótica ativada em resposta aos
extratos era a via intrínseca, e que a produção de radicais livres estava, pelo menos em
parte, relacionada ao efeito bifásico (apoptose ou necrose) encontrado. Os resultados
aqui apresentados demonstram a existência de uma seletividade do efeito citotóxico,
uma vez que os extratos não induziram morte em culturas de astrócitos na dose em que
foi observada morte por apoptose nos gliomas. Ademais, uma potente atividade
apoptótica foi observada com o composto ativo isolado da esponja marinha alvo desse
estudo. Os nossos resultados sugerem que a esponja marinha alvo desse estudo pode ser
considerada uma boa fonte para o desenvolvimento de novas drogas contra o câncer.
1
ABSTRACT
Over the last years, the marine environment has been screened for a variety of
compounds with different biological activities. Among all marine organisms, sponges
represent one of the most promising source of new drug leads for cancer. Although
Brazil has the second most extensive coastline after Australia, there are just few reports
in which the authors have screened Brazilian sponge extracts for biological activities. In
this work, we examine the anti-proliferative effects of crude extracts of the marine
sponge Polymastia janeirensis in the U138MG human glioma cell line. Moreover, we
investigate the effects on selective cytotoxicity in the glioma cells in comparison with a
normal, untransformed cell culture, as well as examine the apoptotic pathway activated
in response to treatments with extracts of P. janeirensis. Yet, we performed a bioassay-
guided fractionation to found the active fractions in the extracts. Exposure of glioma
cells to treatments resulted in cell number decrease with both aqueous and organic
extracts. Moreover, sponge extracts reduced glioma cell viability. However, higher
doses (50 e 100 µg/ml) induced a stronger cytotoxic effect when compared to the lower
dose tested (10 µg/ml), inhibiting more than 80% of cellular growth and viability. Our
results showed that sponge extracts caused necrosis in the glioma cell line at higher
doses, while a high percentage of apoptotic glioma cells were observed at 10 µg/ml. We
also demonstrated that marine sponge extracts induces oxidative cell death through a
receptor-independent pathway under our assays condictions, and ROS productions may
be related to the biphasic effect (apoptosis or necrosis) observed. Furthermore, our
results suggested a selective cytotoxic effect on glioma cell line compared to a normal
cell culture, since the effect on viability found in glioma cells was not observed in
astrocyte cultures with the lower dose (10 µg/ml). Yet, a strong apoptotic activity was
observed with the active fraction isolated from P. janeirensis. Our results suggest that
this marine sponge may be considered a good source for new antiglioma metabolites.
2
LISTA DE ABREVIATURAS
U138MG - linhagem de glioma humano U138MG
TMZ - Temozolomida
Z-VAD - inibidor de caspases
Z-LEHD - inibidor específico de caspase-9
Z-IETD - inibidor específico de caspase-8
HPLC - high performance liquid chromatography
PDA detector - detector de arranjo de fotodiodos
ROS - espécies reativas de oxigênio
DCFH - diclorohidrofluoresceína
PI - iodeto de propídeo
PARP - poly-ADP-ribose polymerase
3
LISTA DE FIGURAS
Figura 1. Foto in situ da esponja marinha P. janeirensis...............................................15
4
APRESENTAÇÃO
Essa Tese sugere que a esponja marinha Polymastia janeirensis pode ser
considerada uma boa fonte para o desenvolvimento de novas drogas contra o câncer. Os
“Materiais e Métodos” e os “Resultados” estão apresentados na forma de artigos
científicos. O primeiro artigo descreve um efeito citotóxico seletivo de diferentes
extratos da esponja marinha em uma linhagem de glioma humano (U138MG), e foi
aceito para publicação no periódico Investigational New Drugs, um dos mais
importantes na área do desenvolvimento de novas drogas contra o câncer. O segundo
artigo descreve o mecanismo de morte ativado em resposta aos extratos, bem como o
envolvimento de espécies reativas de oxigênio nos efeitos encontrados. Esse artigo foi
submetido para publicação no periódico Toxicology in Vitro. O terceiro trabalho
descreve o isolamento da fração ativa responsável pela atividade antiproliferativa
encontrada nos extratos, tendo sido esse submetido para publicação, como “Rapid
Communication”, no periódico The Biological Bulletin.
A “Discussão” acerca dos resultados obtidos nessa Tese contém uma ampla
interpretação dos mesmos, além de importantes considerações no que se refere aos
resultados encontrados na literatura específica.
As “Referências Bibliográficas” se referem àquelas apresentadas na
“Introdução” e na “Discussão” dessa Tese. Por fim, são apresentadas as conclusões e as
perspectivas geradas com esse trabalho.
5
I - INTRODUÇÃO
6
I. INTRODUÇÃO
I. 1 Produtos naturais de origem marinha
I. 1. 1 Um breve histórico
Nas últimas décadas, a bioquímica ecológica dos organismos marinhos tem
emergido como uma interessante área a ser pesquisada. Os oceanos são fontes de um
grande grupo de produtos naturais, e a descrição de novos compostos, muitas vezes
únicos em suas estruturas, tem superado todas as expectativas (Faulkner, 2000). Esses
potentes compostos marinhos são utilizados como defesa química contra predadores e
parasitas, incluindo bactérias e fungos, mas também são conhecidos por modularem
diversos processos biológicos (Pawlik et al., 1993; Proksch et al., 1994; Ebel et al.,
1997).
Os primeiros estudos com compostos de origem marinha remetem ao início do
século XX, com o isolamento de alguns esteróides de esponjas e de outros animais, de
carotenóides presentes em diversos invertebrados marinhos, além de alguns estudos
com toxinas encontradas em peixes da família Tetraodontideae (Doré, 1990; Henze,
1909; Lederer, 1938; Tahara, 1910). Todavia, foi somente a partir de 1950 que a
atenção voltada à fauna marinha teve seu início. Com um trabalho pioneiro realizado
por Bergman e seus colaboradores, foram isolados diversos arabino-nucleosídeos da
esponja Cryptotethya crypta (Bergman e Feeney, 1950). Essa importante descoberta
levou à síntese de análogos sintéticos como o Ara C e o Ara A (Kijjoa & Sawangwong,
2004). O Ara-C, atualmente vendido pela Pharmacia & Upjohn Company sob o nome
de Cytosar-U
®
, é um composto utilizado na terapia contra o câncer (Newman & Cragg,
2004). Já o composto Ara A, vendido sob o nome de Vidarabine
®
ou Vidarabin Thilo
®
,
possui atividade antiviral (Kijjoa & Sawangwong, 2004). Além disso, outros
7
nucleosídeos foram obtidos a partir desses estudos, como o acyclovir e o AZT (Munro
et al., 1993; Newman et al., 2000; Pettit et al., 2001).
I. 1. 2 Esponjas marinhas como fontes de novos fármacos
As esponjas (Filo Porifera) constituem um importante grupo no Reino Animal,
sendo um modelo essencial na transição entre os organismos unicelulares e
pluricelulares. Esses animais surgiram há mais de 550 milhões de anos, no período Pré-
Cambriano, Era Paleozóica. No Cambriano Inferior já existiam representantes de todos
os grupos encontrados recentemente (Bergquist, 1978) e aproximadamente 7.000
espécies das 15.000 estimadas, já foram descritas pelos pesquisadores (Hooper and
Lévi, 1994). Aproximadamente 99% das espécies de esponjas ocorrem no ambiente
marinho, e somente 1% das espécies encontra-se em ambientes de água doce.
O nome “Porifera” é justificado pela presença de pequenas aberturas na
superfície, chamadas poros. Cada poro é formado por um porócito, uma célula em
forma de anel que se estende da superfície externa até a cavidade central denominada
átrio ou espongiocele. Internamente, a parede do corpo é revestida pelos coanócitos, que
são células flageladas típicas dos poríferos. Os coanócitos promovem a filtração da água
capturando microorganismos e partículas alimentares nela presentes. Após a filtração, a
água é expelida para o meio externo através de uma abertura maior chamada ósculo. A
parede corporal é relativamente simples, com a superfície externa revestida de células
achatadas conhecidas como pinacócitos, que constituem a pinacoderme. Os pinacócitos
secretam um material que fixa a esponja ao substrato. Por baixo da pinacoderme está o
mesohilo, que consiste em uma matriz protéica gelatinosa contendo o material
esquelético e células amebóides. O mesohilo é equivalente ao tecido conjuntivo dos
outros metazoários. O esqueleto é relativamente complexo e proporciona uma estrutura
8
de sustentação para as células vivas do animal. A maioria das esponjas tem esqueleto
formado por fibras de espongina juntamente com estruturas chamadas espículas,
parecidas com pequenas agulhas cristalinas de sílica ou de carbonato de cálcio. O
esqueleto que sustenta as esponjas é constituído por uma rede de espículas rígidas,
fibras flexíveis e sedimentos externos, como areia. A combinação das dimensões, do
tipo e da distribuição das espículas, bem como sua relação com o esqueleto fibroso, é a
principal ferramenta utilizada para identificar esponjas.
As esponjas são organismos filtradores capazes de ingerir partículas de tamanho
entre 5-50 μm através de células do mesohilo e da pinacoderme, e micropartículas de
0,3 a 1 μm pelas câmaras de coanócitos (Simpson, 1984). Um espécime da pequena
esponja silicosa de 1 kg, Geodia cydonium, filtra 24.000 litros de água por dia (Vogel,
1977). Sésseis quando adultos, sua distribuição está condicionada à duração de seu
curto período larval livre natante, em geral de poucas horas.
Devido a sua condição séssil e filtradora, as esponjas marinhas desenvolveram,
ao longo da evolução, um efetivo sistema contra o estresse ambiental, produzindo
toxinas e outros compostos para repelir e deter predadores (Uriz et al., 1996; Pawlik et
al., 2002), para competir por espaço com outras espécies sésseis (Porter and Targett,
1998; Davis et al., 1991; Becerro et al., 1997), bem como para comunicação e proteção
contra infecção. Quando comparadas a outros organismos marinhos, as esponjas
possuem a maior taxa de compostos citotóxicos, com mais de 10% das espécies
investigadas apresentando alguma atividade biológica (Osinga et al., 1998; Zhang et al.,
2003). Além disso, dos 15.000 produtos marinhos descritos, as esponjas respondem por
mais de 5.300 desses, e, a cada ano, centenas de novos compostos de origem marinha
estão sendo descobertos. A considerável diversidade química desses animais
compreende além de nucleosídeos incomuns, terpenos, esteróis, peptídios cíclicos,
9
alcalóides, ácidos graxos, até peróxidos e derivados de aminoácidos halogenados
(Sipkema et al., 2005).
Extratos naturais contêm uma excepcional diversidade de moléculas e continuam
sendo uma das fontes mais promissoras para o desenvolvimento de novos fármacos
(Newman et al., 2003). Ademais, estudos realizados pelo National Cancer Institute dos
EUA, retratam as esponjas marinhas como os organismos que mais produzem
moléculas de alta singularidade com possível interesse farmacológico e potencial
utilização no tratamento de doenças como o câncer, a AIDS e as leucemias (Kelecom et
al., 1991). Neste contexto, extratos de diferentes espécies de esponjas marinhas têm sido
examinados em várias linhagens tumorais celulares, bem como em modelos animais
(Ahond et al., 1988; Burres et al., 1991; Fusetani et al., 1992; Gunasekera et al., 1991;
Kobayashi et al., 1989; Perry et al., 1988; Sakai et al., 1986; ter Haar et al., 1996).
Trabalhos com a utilização de extratos brutos de esponjas marinhas também têm
demonstrado propriedades antimicrobianas, antifúngicas e antiinflamatórias para
algumas espécies da região do Caribe, Mediterrâneo, Indo-Pacífico e Atlântico Oriental.
Entre os diferentes compostos isolados até hoje, a Psammaplin A, por exemplo,
possui propriedades anti-câncer. Da mesma forma, uma relevante atividade antiviral em
HIV-1 foi observada em algumas avaronas derivadas da esponja Dysidea cinerea
(Hirsch et al., 1991). Um outro trabalho demonstrou a inibição de CDKs, GSK-3β e
CK1 por hymenialdisine (HD), um composto isolado de várias espécies de esponjas
marinhas, sugerindo que esta molécula possa contribuir nos estudos de doenças
neurodegenerativas, como o Alzheimer e o Parkinson (Meijer et al., 2000).
Alguns compostos citados anteriormente já estão sendo investigados ou mesmo
sendo desenvolvidos como novos fármacos (Faulkner, 2002). O princípio
antiinflamatório manoalida de Luffariella variabilis já está disponível no mercado
10
(Monks et al., 2002). Em estudos pré-clínicos de Fase I encontram-se os compostos
KRN7000 de Agelas mauritianus e IPL 576092 de Petrosia contignata e, em Fase II, o
agente anti-câncer halicondrina B de Halichondria okadai.
Entretanto, um dos principais problemas que a indústria farmacêutica tem
enfrentado no desenvolvimento de programas desse gênero é a dificuldade de obtenção
do material biológico, tendo em vista as altas profundidades onde as esponjas em
questão se encontram, além do fato do baixo rendimento dos compostos em análise;
problemas esses diretamente relacionados a fatores ambientais e geográficos. Mesmo
assim, apesar da recente literatura com essa abordagem, a existência de programas de
pesquisa para a proteção de esponjas em locais como o Caribe e a Austrália acaba por
refletir o maior número de trabalhos científicos com espécimes dessas regiões.
I. 1. 3 Potencial de esponjas marinhas brasileiras
O Brasil possui aproximadamente 8.000 km de costa litorânea (a segunda maior
do mundo depois da Austrália), sendo um dos países com o maior índice de
biodiversidade do mundo. Entretanto, poucos trabalhos explorando a diversidade
química da nossa fauna marinha foram realizados, uma vez que o principal foco da
química de produtos naturais brasileira foi direcionado, por muitos anos, ao estudo de
plantas medicinais.
Características peculiares como fatores ambientais (exposição à alta intensidade
de luz e altas pressões de oxigênio) e uma geografia propícia (águas rasas) fazem das
esponjas do nosso litoral candidatas ideais para a obtenção de novos fármacos,
tornando-se relevante a pesquisa sobre a aplicação dos mesmos no tratamento de
doenças de alto impacto sócio-econômico. Sendo assim, a utilização de produtos
naturais extraídos de esponjas marinhas brasileiras pode ser considerada uma importante
11
ferramenta para o desenvolvimento de novos medicamentos; alvo principal da indústria
farmacêutica. Ademais, é importante destacar que o crescente estudo de propriedades
farmacológicas em esponjas marinhas em outros países contribuiu para a criação de
programas de preservação desses animais, bem como do eco-sistema em que eles
vivem, e que a existência de programas de pesquisa com esponjas nessas regiões reflete
o maior número de moléculas e patentes obtidas nesses países.
No que diz respeito à pesquisa com esponjas marinhas no Brasil, a maior parte
dos trabalhos tinha como objetivo principal o inventariamento faunístico e o estudo
taxonômico desses animais. Entretanto, um considerável aumento no número de
trabalhos explorando as propriedades farmacológicas de esponjas coletadas no litoral
brasileiro pode ser observado nos últimos anos. Entre as atividades encontradas nos
extratos analisados, destacam-se atividades antimicrobianas (Muricy et al., 1993),
citotóxica e neurotóxica (Rangel et al., 2001), anti-câncer (Monks et al., 2002) e
desorganização de microtúbulos (Prado et al., 2004). Além disso, uma importante
revisão sobre produtos naturais marinhos oriundos de organismos coletados no Brasil
pode ser encontrada em Berlinck et al., 2004.
I. 2 Biologia dos gliomas
De acordo com a Organização Mundial de Saúde (OMS), a cada ano surgem
nove milhões de novos casos de câncer no mundo. No Brasil, segundo o Ministério da
Saúde, mais de quinhentos mil novos casos de câncer surgem por ano, e
aproximadamente 100 mil pessoas morrem anualmente (www.saude.gov.br). Contudo,
apesar da variedade de fármacos existentes para o tratamento de diferentes tipos de
câncer, problemas referentes à baixa seletividade dos antineoplásicos e ao fenótipo de
12
resistência a múltiplas drogas dão sustentação para a busca por novas moléculas com
propriedades antiproliferativas.
De particular interesse são os gliomas. Esse tipo de neoplasia primária ocorre
principalmente no cérebro, podendo afetar também outras partes do sistema nervoso
central, como a medula espinhal e os nervos ópticos. São tumores que se originam de
células da linhagem astrocítica, possuindo um alto grau de malignidade e, apesar do
tratamento agressivo com radioterapia e quimioterapia, a sobrevida costuma ser inferior
a 12 meses. Na maioria dos casos, essas neoplasias se originam de mutações
espontâneas em genes que controlam o ciclo celular ou a divisão celular. Além disso, os
gliomas são caracterizados por alterações em suas rotas apoptóticas (Ziegler et al.,
2008). Desta maneira, adquirem uma resistência intrínseca a esse mecanismo de morte
celular, dificultando ainda mais o tratamento.
Um mecanismo de morte celular em resposta a vários agentes quimioterápicos,
incluindo agentes alquilantes, é a morte celular programada, também chamada de
apoptose. Em contraste à morte celular por necrose, a apoptose é fisiologicamente mais
vantajosa, uma vez que as células mortas são fagocitadas antes da lise celular e da
liberação de mediadores pró-inflamatórios (Shacter et al., 2000; Anderson et al., 2002;
Anderson et al., 2003; Fadok and Henson, 2003; Lauber et al., 2004). Nesse sentido, um
recente estudo demonstrou que Temozolomide (um novo agente alquilante utilizado na
terapia contra gliomas) é capaz de induzir apoptose em uma linhagem de glioblastoma
humano (U87MG) (Arabinda et al., 2004). Ademais, o uso desse novo fármaco tem
aumentado a sobrevida dos pacientes em dois anos quando administrado durante e após
a radioterapia (Stupp et al., 2005).
13
I. 3 Radicais livres e morte celular programada
Um aspecto fundamental do metabolismo aeróbico é a geração de espécies
parcialmente reduzidas de oxigênio molecular, conhecidas como espécies reativas de
oxigênio ou radicais livres. Sob condições fisiológicas, a fosforilação oxidativa é a
principal fonte endógena desses radicais.
Diversos processos biológicos são modulados por radicais livres. Entretanto,
quando existe um desequilíbrio entre a produção de espécies reativas de oxigênio e os
mecanismos de defesas antioxidantes, ocorre um progressivo acúmulo de danos em
biomoléculas (lipídios, proteínas e DNA), levando a célula à morte. Além disso, sabe-se
que o excesso de radicais livres pode causar morte celular, mas o mecanismo de morte
(necrose ou apoptose) depende, entre outros fatores, do tipo e da concentração de
radicais produzidos no ambiente celular.
Apoptose, ou morte celular programada, é um complexo processo que requere a
integração de diferentes sinais intracelulares, sendo um dos principais mecanismos
pelos quais diferentes drogas citotóxicas matam células tumorais. Esse mecanismo de
morte ocorre principalmente por duas rotas referidas como extrínseca ou intrínseca,
sendo a primeira dependente da ligação de ligantes nos chamados receptores de morte
encontrados na membrana celular, e a segunda, da liberação de sinais de morte da
mitocôndria para o citoplasma. Ambas as rotas acabam convergindo e ativando a
caspase-3, uma caspase executora que cliva proteínas intracelulares e causa morte
celular.
Recentes trabalhos com produtos naturais de origem marinha têm demonstrado
uma correlação entre a produção de radicais livres e a morte celular por apoptose.
Entretanto, os mecanismos envolvidos nessa ativação, além da rota ativada (extrínseca
ou intrínseca), ainda não são conhecidos.
14
I. 4 A esponja marinha Polymastia janeirensis
A esponja marinha P. janeirensis (Boury-Esnault, 1973), ordem Hadromerida,
família Polymastiidae, apresenta uma coloração que pode variar entre tons de marrom,
na sua parte incrustante, até uma intensa coloração marrom, azul ou laranja, nas suas
projeções cilíndricas, podendo ser encontrada desde a zona entre-marés, até 39 m de
profundidade (Figura 1).
Figura 1: Foto in situ da esponja marinha P. janeirensis.
Embora defesas químicas não possam ser diretamente equacionadas com
potentes propriedades farmacológicas, é impressionante como se correlacionam na
realidade. Isso é particularmente importante para a esponja P. janeirensis. Conforme
observações de campo, essa esponja apresenta uma aparente falta de predadores
naturais. Além disso, ao serem cortadas, no momento da coleta, ocorre a liberação de
um líquido laranja com propriedades antimicrobianas na água. Entretanto, poucas
15
informações são conhecidas sobre essa espécie, e somente dois trabalhos descrevem
propriedades biológicas em extratos de P. janeirensis. No primeiro (Monks et al., 2002),
foi observado um efeito citotóxico em extratos orgânicos contra três linhagens de câncer
(HT-29, U-373 e NCI-H460), com um IC
50
entre 50 e 100 µg/ml. Além disso, extratos
aquosos retardaram significativamente a migração de leucócitos em um ensaio
quimiotático. Contudo, o modo de ação dessas atividades biológicas permanece
desconhecido. Já no outro trabalho (da Silva et al., 2006), uma importante atividade
antiviral foi observada em extrato aquoso contra o rotavírus RV-SA11. Nesse caso, o
extrato inibia os últimos estágios de replicação do rotavírus.
Os únicos compostos isolados a partir de esponjas do gênero Polymastia
encontrados na literatura são esteróis e tetrahidroxiamidas oriundos da esponja P. tenax,
coletada na Colômbia. Esses compostos exibiram significante atividade citotóxica
contra linhagens de carcinoma humano de pulmão (A-549), carcinoma humano de cólon
(HT-29 e H-116) e carcinoma humano de próstata (Faulkner, 2001; Santafe et al.,
2002). No entanto, os mecanismos envolvidos no processo antiproliferativo
permanecem desconhecidos.
16
II - OBJETIVOS
17
II. OBJETIVOS
Extratos de esponjas marinhas possuem uma excepcional diversidade de
moléculas, sendo uma das fontes mais promissoras para o desenvolvimento de novas
drogas contra o câncer. Portanto, os objetivos desta Tese são:
1- Examinar o efeito antiproliferativo de extratos brutos da esponja marinha P.
janeirensis coletada no litoral de Santa Catarina, Brasil, utilizando como modelo de
estudo uma linhagem de glioma humano (U138MG);
2- Testar a seletividade dos extratos, utilizando uma cultura de células não
transformadas;
3- Examinar a produção de radicais livres, o mecanismo de morte e as rotas ativadas;
4- Isolar, dos extratos da esponja marinha P. janeirensis, a fração ativa responsável pela
atividade antiproliferativa encontrada.
18
III - ARTIGOS CIENTÍFICOS
19
III. 1 - Artigo 1
Brazilian marine sponge Polymastia janeirensis induces apoptotic cell death in
human U138MG glioma cell line, but not in a normal cell culture
Investigational New Drugs, 2008 (in press)
20
PRECLINICAL STUDIES
Brazilian marine sponge Polymastia janeirensis induces
apoptotic cell death in human U138MG glioma cell line,
but not in a normal cell culture
Mario Luiz Conte da Frota Jr & Elizandra Braganhol &
Andrés Delgado Canedo & Fabio Klamt &
Miriam Anders Apel & Beatriz Mothes & Cléa Lerner &
Ana Maria Oliveira Battastini &
Amélia Teresinha Henriques &
José Cláudio Fonseca Moreira
Received: 14 January 2008 / Accepted: 19 March 2008
#
Springer Science + Business Media, LLC 2008
Summary Marine sponges have been prominently featured
in the area of cancer research. Here, we examined the anti-
proliferative effects of crude extracts (aqueous and organic)
of the Brazilian marine sponge Polymastia janeirensis in
the U138MG human glioma cell line. Moreover, we
examined the effects of extra cts on selective cytotoxicity
in the glioma cells in comparison with a normal cell culture.
Exposure of glioma cells to treatments (24 h) resulted in cell
number decrease at all doses tested, with both aqueous and
organic extracts (IC
50
<20 and <30 μg/ml, respectively).
Parallel to this result, sponge extracts reduced glioma cell
viability (IC
50
<15 μg/ml for both extracts). However,
higher doses (50 and 100 μg/ml) induced a stronger
cytotoxic effect when compared to the lower dose tested
(10 μg/ml), inhibiting more than 80% of cellular growth
and viability. Propidium iodide uptake and flow cytometry
analysis further showed that sponge extracts caused
necrosis in the glioma cell line at higher doses, while a
high percentage of apoptotic glioma cells were observed at
10 μg/ml. Moreover, apoptosis was prevented by the pan-
caspase inhibitor Z-VAD, suggesting that marine sponge
extracts, at lower doses, induce caspase-dependent apopto-
sis in U138MG glioma cells. Surprisingly the extracts
herein tested were more effective than temozolomide, a
potent inductor of apoptosis used for the treatment of
malignant gliomas. Furthermore, our results suggested a
selectivity cytotoxic effect on glioma cell line in compar-
ison with a normal cell culture, since the effect on viability
found in glioma cells was not observed in astrocyte cultures
with the lower dose (10 μg/ml). Thus, this marine sponge
may be considered a good candidate for development of
new cancer medicines with antitumor activity against
gliomas.
Keywords Cancer
.
Glioma cells
.
Apoptosis
.
Marine sponges
.
Polymastia janeirensis
.
New drugs
Invest New Drugs
DOI 10.1007/s10637-008-9134-3
M. L. C. da Frota Jr
:
F. Klamt
:
J. C. F. Moreira
Centro de Estudos em Estresse Oxidativo (CEEO), ICBS,
UFRGS,
Porto Alegre, Brazil
E. Braganhol
:
A. M. O. Battastini
Laboratório de Enzimologia, ICBS, UFRGS,
Porto Alegre, Brazil
A. D. Canedo
Laboratório de Cardiologia Molecular e Celular, IC/FUC,
Porto Alegre, Brazil
M. A. Apel
:
A. T. Henriques
Faculdade de Farmácia, UFRGS,
Porto Alegre, Brazil
B. Mothes
:
C. Lerner
Fundação Zoobotânica, Museu de Ciências Naturais,
Porto Alegre, Brazil
M. L. C. da Frota Jr (*)
Departamento de Bioquímica, ICBS, UFRGS,
Rua Ramiro Barcelos 2600-ANEXO,
Porto Alegre 90035-003 RS, Brazil
e-mail: [email protected]
21
Introduction
Cancer is considered a public health problem in developed
and in developing countries. In Brazil, it is estimated that
100,000 people die annually [1]. Of particular interest are
the mal ignant gliomas. These tumors arise from cells of the
astrocytic lineage, and are considered the most devastating
primary tumors in the brain, representing 50–60% of this
type of tumor. As a result of high proliferation, invasivity,
and resistance to radiation [2], the prognosis for patients
with malignant gliomas is poor and the mean survival is
less than 12 months [3 ].
Despite the intense efforts to develop treatments,
effective agents are still not available. Therefore, it is of
seminal importance to provide new drug leads that may be
developed into new cancer medicines with antitumor
activity against gliomas. In this regard, natural products
extracts continue to be the most promising source of new
drug leads for cancer. They contain an exceptional diversity
of chemotypes, suitable for high throughput screening and
further development by combinatorial synthesis, molecular
modeling, and structure versus activity studies [4].
Over the last decades, the marine environment has been
screened for a variety of compounds with different
biological activities. Among all marine organisms, sponges
represent one of the most promising source of marine
bioactive compounds particula rly for pharmaceutical leads
[5, 6]. In order to purify new active compounds with
biological activities and potential application in biomedi-
cine, extra cts from marine sponges around the world have
been examined for antineoplastic activity in various tumor
cell lines, as well as in animal models [7–14]. At least two
drugs (Ara-A and Ara-C) synthetically derived from sponge
metabolites have been clinically used in long-term cancer
treatments [15]. Furthermore, sponges also have been
screened for a variety of other biological activities, e.g.,
antimicrobial, haemolytic, hemagglutinating, ichthyotoxic,
lethal properties and others [16–18]. However, there are just
few reports in which the authors have screened Brazilian
sponge extracts for biological activities. To date, only
limited screening evaluations of extracts of Brazilian
marine sponges have been reported [19–22 ].
In this report, we examine the anti-proliferative effects of
crude extracts (aqueou s and organic) of the Brazilian
marine sponge Polymastia janeirensis in the U138 MG
human glioma cell line. Moreover, we examine the effects
of extracts on selective cytotoxicity in the glioma cell line
in comparison with a normal, untransformed cell culture.
This study is part of a collaborative program among several
Brazilian institutions (Centro de Estudos em Estresse Oxida-
tivo, Universidade Federal do Rio Grande do Sul; Fundação
Zoobotânica do Rio Grande do Sul, Museu de Ciências
Naturais; Departamento de Bioquímica, Universidade Federal
doRioGrandedoSul;andFaculdadedeFarmácia,
Universidade Federal do Rio Grande do Sul) for the collection
and screening of Brazilian marine sponges for biological
activities, with the aim of identifying new sponges species and
novel molecules with promising and potentially useful
therapeutic activities.
Materials and methods
Sponge sampling, identification and extract preparation
Sponges samples were collected manually from exposed
and semi-exposed habitats, at depths of between 0.5 and
20 m, from locations on the coastline of the Estate of Santa
Catarina (southern Brazil). Taxonomic designation was
based on scanning electron microscope studies and on
skeletal slides and dissociated spicule mounts. Specimens
of all materials are dep osited in the Museu de Ciências
Naturais-Porifera collection of the Fundação Zoobotânica
do Rio Grande do Sul, Brazil. Aqueous and organic extracts
were obtained as previously described [20].
Cell cultures
U138MG human glioblastoma cell line was obtained from
the American Type Culture Collection (Rockville, Mary-
land, USA). Cells were grown in culture flasks in
Dulbecco’s modified Eagle’s medium (DMEM)/15% fetal
bovine serum (FBS) (v/
v) (Cultilab, Campinas, SP, Brazil)
and seeded in 24-well plates (TTP plates) at densities of 1×
10
4
cells/well in 500 μl medium per well. Culture cells
were maintained in 5% CO
2
/95% air at 37 °C and allowed
to grow to confluence.
Primary astrocyte cultures were prepared as previously
described [23]. Briefly, hippocampus, cortex and cerebel-
lum of newborn Wistar rats (1–2 days old) were removed,
and dissociated mechanically in a Ca
2+
- and Mg
2+
-free
balanced salt solution pH 7.4, containing 137 mM NaCl,
5.36 mM KCl, 0.27 mM Na
2
HPO
4
, 1.1 mM KH
2
PO
4
,
6.1 mM glucose. After centrifugation at 1,000 rpm for 5 min
the pellet was resuspended in culture medium (pH 7.6)
containing 1% DMEM, 8.39 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), (pH 7.6), 23.8 mM
NaHCO3, 0.1% fungizone, 0.032% garamicine and 10%
fetal calf serum from Gibco. The cells were plated at a
density of 1.5×10
5
cells cm
−2
onto 24 multiwell plates pre-
treated with poly-
L-lysine. Cultures were maintained in 5%
CO
2
/ 95% air at 37 °C and allowed to grow to confluence
and used at 21–28 days in vitro. Medium was changed every
3–4 days. Immunocyto chemical studies (not shown)
revealed that >95% of the cells in our cultures stained for
glial fibrillary acidic protein.
Invest New Drugs
22
Treatments
Immediately before experiments, both the aqueous and
organic extracts were dissolved in water and dimethyl
sulfoxide (DMSO), respectively, at a concentration of
1mg/ml(w/v). The amount o f DMSO (maximum 0.25%)
was p roven not to affect the experimen ts. The final
concentrations of the extracts tested ranged from 10 to
100 μg/ml. After reaching subconfluence, the cultures
were exposed to sponge extracts for 24 h. Control cultures
were performed with D MSO (0.25% final concentr atio n)
in the absence of extracts.
Cell counting
At the end of the treatment (24 h), the medium was
removed, cells were washed with phosphate-buffered saline
(PBS) and 200 μl of 0.25% trypsin/ethylenediaminetetra-
acetic acid (EDTA) was added to detach the cells, which
were counted in a hemocytometer.
Assessment of glioma cell viability
Following treatments, cell viability was assessed by the 3-
(4,5-dimethyl)-2,5 diphenyl tetrazolium brom ide (MTT)
assay. This method is based on the ability of viabl e cells
to reduce MTT and form a blue formazan product. MT T
solution (sterile stock solution of 5 mg/ml) was added to the
incubation medium in the wells at a final concentration of
0.5 mg/ml. The cells were left for 60 min at 37 °C in a
humidified 5% CO
2
atmosphere. The medium was then
removed and plates were shaken with DMSO for 30 min.
The optical density of each well was measured at 550 nm
(test) and 690 nm (reference). Results were expressed as the
percentage of cell viability against the control.
Propidium iodide assay
Cellular damage was assessed by fluorescent image
analysis of propidium iodide (PI) uptake. PI is excluded
from healthy cells, but following loss of membrane
integrity enters cells, binds to DNA and becomes highly
fluorescent. At the end of the treatment, glioma cells were
incubated with PI (7.5 μ mol/l) for 1 h. PI fluorescence was
excited at 515–560 nm using an inverted microscope
(Nikon Eclipse TE300; Nikon Inc., Melville, New York,
USA) fitted with a standard rhodamine filter and a
minimum of 200 cells was counted per treatment in four
different fields. The presence of stained, enlarged nuclei
with normal structure was scored as a necrotic cell and the
results were expressed as a ratio of PI labeled cells/total
number cells. Images were captured using a digital camera
connected to the microscope.
Flow cytometry analysis
For annexin V/PI (AV/PI) staining, treated glioma cells (1×
10
6
) were washed twice in PBS (137 mM NaCl, 2.7 mM
KCl, 4.3 mM Na
2
HPO
4
.2H
2
O, 1.4 mM KH
2
PO
4
, pH 7.4)
containing 1 mM EDTA (PBS-EDTA) and subsequently
trypsinized with 0.13 g/L trypsin in PBS-EDTA. Trypsin
was inhi bited with bovine foetal serum 10%, and medium
washes, and cells were combined and centrifuged (5 min,
200×g, 4 °C). Cells were allowed to recover from
trypsinization in complete medium (30 min, 37 °C).
Externalized phosphatidylserine was labeled (15 min, 0 °C)
with 5 μl fluorescence (FITC)-conjugated annexin V in
80 μl binding buffer (10 mM HEPES pH 7.4, 145 mM
NaCl, 5 mM KCl, 1.0 mM MgCl
2
.6H
2
O, 1.8 m M
CaCl
2
.2H
2
O). Propidium iodide 2 μM was added 10 min
prior to analysis on a FACScalibur flow cytometer (BD
PharMingen). When green fluorescence was plotted against
red fluorescence (PI), distinct cell populations could be
detected: viable cells (FITC−/PI−), apoptotic cells (FITC+/
PI− and FITC+/PI+), and necrotic cells (FITC−/PI+), as
previously described [24]. Ten thousand cells were ana-
lyzed per sample, and data were reported as the percentage
of apoptotic cells and necrotic cells. Temozolomide (3,4-
dihydro-3 -methyl-4-oxoimi dazo-(5,1-d)-1,2,3,5-tetrazin-8-
carboxamide, TMZ), a cytotoxic alkylating agent [25], was
used as a control of cell death.
Statistical analysis
Results were expressed as the mean±SEM of at least three
independent experiments. Data were analyzed by a one-way
analysis of variance (ANOVA), using a Newman Keuls test
to compare mean values across groups. When appropriate,
Student’s t-test was performed. Differences were consi dered
to be significant when p<0.05. Dose response curves were
plotted, and the IC
50
values (concentrations at which
cellular effects are inhibited by 50%) were calculated using
non-linear regression analysis.
Results
Marine sponge extracts decreased U138MG glioma cell
growth and viability
In order to investigate the effect of marine sponge extracts
on proliferation/viability of U138MG glioma cell line, cell
cultures were treated with extracts, the cell number was
counted and a MTT assay was performed. Exposure of
glioma cells to treatments for 24 h resulted in cell number
decrease at all doses tested, with both aqueous and organic
extracts (Fig. 1), w ith an IC
50
<20 and <30 μg/ml,
Invest New Drugs
23
respectively. However, we observed that higher doses (50
and 100 μg/ml) of both extracts have stronger cytotoxic
effects, inhibiting more than 80% of glioma cellular growth.
Parallel to this result, sponge extracts treatments also
resulted in a reduction of glioma cell viability, as evidenced
by a diminished ability of glioma cells to reduce MTT, with
an IC
50
<15 μg/ml for both aqueous and organic extracts
(Fig. 2). Moreover, similar to cell counting, higher doses
also had stronger cytotoxic effects. The reduction in MTT
staining suggests not only glioma cell damage, but also a
decrease in cell proliferation when compared with the
control.
Cell death induced by marine sponge extracts
To determine whether the su ppression of g lioma cell
proliferation was due to the induction of necrosis, glioma
cells were treated for 24 h and then analyzed for cell
membrane permeability by PI. As shown in Fig. 3,PI
incorporation was higher in cells treated with both aqueous
and organic extracts at 50 and 100 μg/ml, suggesting that
these doses caused a loss of membrane integrity, which is an
indication of cell death by necrosis. However, at 10 μg/ml,
this cell death pattern was not observed.
Since higher doses decreased cell viability and induced
necrosis, and the lower dose did not induce necrosis, we
decided to investigate whether the decrease of glioma cell
viability at 10 μg/ml was due to the induction of apoptosis.
Glioma cells then were pre-incubated with membrane-perme-
able pan-caspase inhibitor (N-benzyloxycarbonyl-valyl-alanyl-
aspartyl-fluoromethylketone, Z-VAD-fmk) for 1 h before
treatment with extracts. At the end of treatment, Annexin
V-FITC-bound phosphatidylserine and red fluorescence of
DNA-bound PI in individual cells were measured using a
flow cytometer as described on “Materials and methods”.
As shown in Fig. 4,10μg/ml of both aqueous and organic
extracts resulted in a higher incidence of cell death that was
almost entirely apoptotic. Furthe rmore , cell death was
prevented by the pan-caspase inhibitor Z-VAD, suggesting
that marine sponge extracts, at lower dose, induces caspase-
dependent apoptosis in U138MG glioma cells. It is
noteworthy that TMZ (100 μM), a new cytotoxic alkylating
agent used in therapy for malignant gliomas and a potent
inhibitor of cell growth and angiogenesis at non-toxic doses
was less effective than marine sponge extracts under our
assay conditions.
Selective cytotoxic effect of marine sponge extracts
on U138MG glioma cells
To evaluate the selective cytotoxic effect of marine sponge
extracts of P. janeirensis on glioma cell cultures, primary
astrocyte cultures were treated for 24 h and an MTT assay
was performed. Exposure of astrocyte cultures to treatments
resulted in a decrease in cell viability at concentrati ons of
50 and 100 μg/ml (>90% for both aqueous and organic
extracts), as evidenced by a decreased ability of astrocyte
cells to reduce MTT. However, treatment with 10 μg/ml of
aqueous and organic extracts did not decrease cell viability
in astrocyte cultures, suggesting that this dose decreases
cell viability only in glioma cells. Interestingly, 10 μg/ml of
both aqueous and organic extracts induced apoptotic cell
death in glioma cells, while 50 and 100 μg/ml induced only
necrosis (Fig. 5).
Discussion
Malignant gliomas occur more frequently than other types
of primary central nervous system tumors. Even with
aggressive treatment using surgery, radiation, and chemo-
therapy, the mean reported survival is less than 1 year [2].
0
20
40
60
80
100
120
Control 10 µg/ml 50 µg/ml 100 µg/ml
Percentage cell viability
Aqueous extracts Or
g
anic extracts
*
*
*
Fig. 2 Effects of marine sponge extracts on U138MG glioma cell
viability. Cell cultures were exposed to extracts (10, 50 or 100 μg/ml)
for 24 h and the cell viability was assessed by the MTT assay as
described in “Material and methods”. Glioma cultures treated with
DMSO (control) was taken as 100% of cell viability. Data are the
means±S.E.M for three individual experiments, and results were
expressed as the percentage of cell viability against the control.
*Statistically different from control, p<0.05 (one-way ANOVA)
0
20
40
60
80
100
120
Control 10 µg/ml 50 µg/ml 100 µg/ml
Percentage cell count of control
Aqueous extracts Or
anic extracts
*
*
*
Fig. 1 Effect of marine sponge extracts on growth of U138MG
glioma cells. Cells were treated for 24 h with extracts (10, 50 or
100 μg/ml) or DMSO (control), and counted in a hemocytometer as
described in “Material and methods”. Cell count in samples treated
with DMSO was considered 100% of cell number. Data are expressed
as means ±S. E.M for three individual experiments. *Statistically
different from control, p <0.05 (one-way ANOVA)
Invest New Drugs
24
Today, current therapy for malignant glioma employs TMZ,
an imidazole tetrazinone whose the methylation of DNA
seems to be the principal mechanism responsible for the
cytotoxicity to malignant cells. Nevertheless, it was clear
from several studies that a significant proportion of tumors
do not respond to TMZ therapy [26–29 ].
In the present study, the ability of crude extracts
(aqueous and organic) of the Brazilian marine sponge P.
janeirensis to inhibit the proliferation of U138MG glioma
cells was examined. Results from cell counting showed that
marine sponge extracts, at all doses tested, inhibited the
proliferation of glioma cells. In addition, the glioma cell
0
5
10
15
20
25
30
35
40
45
50
cell death (%)
Control
TMZ
AE 10 µg/ml
OE 10 µg/ml
Z-VAD + AE
Z-VAD + OE
Necrosis
Apoptosis
Fig. 4 Flow cytometry analysis. Annexin V-FITC-bound phosphati-
dylserine and red fluorescence of DNA-bound PI in individual glioma
cells were measured using a flow cytometer as described under
“Materials and methods”. TMZ temozolomide, AE aqueous extract,
OE organic extracts, Z-VAD pan-caspase inhibitor (100 μM). The data
represent averages from at least three independent experiments carried
out in triplicate
0
20
40
60
80
100
120
Control 10 µg/ml 50 µg/ml 100 µg/ml
Percentage cell viability
Aqueous extracts Or
g
anic extracts
*
*
Fig. 5 Effects of marine sponge extracts on primary astrocyte cultures
viability. Cells were exposed to extracts (10, 50 or 100 μg/ml) for 24 h
and the cell viability was assessed by the MTT assay as described in
“Material and methods”. Astrocyte cultures treated with DMSO
(control) was taken as 100% of cell viability. Data are the means±S.
E.M for three individual experiments, and results were expressed as
the percentage of cell viability against the control. *Statistically
different from control and from10 μg/ml, p<0.05 (one-way ANOVA)
A)
B)
0
0.05
0.1
0.15
0.2
0.25
Ratio PI cells/ total cells
Control 10
µg
/ml 50
µg
/ml 100
µg
/ml
Control
50 µg/ml
100 µg/ml
10 µg/ml
Fig. 3 Representative pictures
(a) and analysis (b)of
U138MG cell cultures stained
with propidium iodide. At the
end of the treatments, the cells
were incubated with PI for 1 h.
After, PI incorporation was
visualized using a Nikon
inverted microscope. The results
were expressed as ratio PI
labeled cells/total number cells
and are representative for aque-
ous extract. Similar effects were
observed in the organic extracts-
treated cells
Invest New Drugs
25
viability was estimated by MTT assay, and our data showed
that extracts of P. janeirensis also reduced the cell viability
of glioma cultures. Nevertheless, we observed that 50 and
100 μg/ml doses were more cytotoxic than 10 μg/ml,
inhibiting more than 80% of cellular growth and cell
viability.
Apoptosis, or programmed cell death, is characterized by
a number of well-defined features which include conden-
sation and fragmentation of the chromatin, internucleoso-
mal DNA cleavage, membrane blebbing, caspase
activation, translocation of phosphatidylserine from the
inner to the outer leaflet of the plasma membrane, and the
ultimate formation of so called “apoptotic bodies” [30]. In
contrast to necrotic cell death, apoptotic cell death is
thought to be physiologically advantageous because the
dying cells are cleared by phagocytosis prior to cell lysis
and release of potentially inflammatory mediators [31–35].
Thus, in order to investigate whether the antiproliferative
effects of the extracts were related to necrotic or apoptotic
cell death, the PI uptake by glioma cells and the flow
cytometry analysis were performed following treatments.
Our results showed that both extracts of sponge were
necrotic in the glioma cell line at higher doses (50 and
100 μg/ml), while a high percentage of apoptotic glioma
cells was observed at 10 μg/ml.
Apoptotic cell death occurs through at least two over-
lapping pathways referred to as extrinsic and intrinsic. The
extrinsic pathway is activated through ligand binding to
death receptors (members of the tumor necrosis factor
receptor superfamily) at the cell surface and activation of
initiator caspase-8. In the intr insic pathway, mitochondria
play a pivotal early role by releasing cell d eath signals into
the cytosol and activating caspase-9 [36]. Both pathways
converge and activate executioner caspase-3, which then
cleaves intracellular protein substrates and causes cell death
[37].
We found that cell death was prevented by the pan-
caspase inhibitor Z-VAD, suggesting that marine sponge
extracts, at lower doses, induce caspase-dependent apopto-
sis in U138MG glioma cells. However, additional studies
are necessary to determine which apoptotic pa thway
(extrinsic or intr insic) was activated in response to treat-
ments. Our preliminary data suggest that marine sponge
extracts cause apoptosis through a receptor-independent
pathway, since cell death was prevented by the caspase-9
inhibitor Z-LEHD, but not by the caspase-8 inhibitor Z-
IETD (data not shown).
Liu et al. (2005) demonstrate d that geoditin A, an
isomalabaricane triterpene isolated from marine sponge
Geodia japonica, induced reactive oxygen species, de-
creased mitochondrial membrane potential and mediated a
caspase-3 ap optosis pathway on human promye locytic
leukemia HL60 cells [38]. However, since caspase-3 is an
effector caspase that converge both pathways, the precise
apoptotic rote activated in this study (receptor-dependent or
receptor-independent) remains unclear. In other work, the
same authors have been demonstrated that stellettin A, also
isolated from G. japonica, induces oxidative cell death
through a Fas
L-caspase-3-apoptotic pathway [39].
One mechanism of tumor cell death in response to
various chemotherapeutic drugs including alkylating agents
[40–42] is programmed cell death or apoptosis. Surprising-
ly, a new cytotoxic alkylating agent used in therapy for
malignant gliomas (TMZ) was less effective than marine
sponge extracts under our assay conditions. In addition, the
cytotoxic effect of marine sponge extracts on primary
astrocyte cultures was evaluated. An important feature of
the present study is that the effect of sponge extracts on
viability found in glioma cell line was not observed in
astrocyte cultures with the lower dose (10 μg/ml), suggest-
ing a selective cytotoxic effect of this dose on U138MG
glioma cell. Interestingly, this is the same concentration that
was ab le to induce apoptosis in the glioma cells, which is
generally believed to be more physiologically beneficial
than necrosis [30–33, 43].
This biphasic effect (apoptosis or necrosis) observed in
glioma cells may be related to the redox state of the cells, as
well as the redox properties of the extracts, and this point
needs further investigation. It is clear that excess levels of
free radicals can cause cell death, but the mode of death
(i.e. necrotic, apoptotic, other) depends on the type,
concentration, source and environment of the oxidant(s)
[44]. Moreover, additional experiments should be per-
formed to elucidate the differences between glioma and
astrocyte cells effects, but one possible explanation is the
distinct signaling pathways in the cells compared here.
Marine natural products have been prominently featured
in the area of cancer research. It is interesting to note that
out of 18 preclinical investigating anticancer agents derived
from marine sources, six are from sponges [45–47],
implying that marine sponges are potential resources for
new anti-cancer agents. However, there are just few reports
in which the authors have screened Brazilian sponge
extracts for biological activities [19–22 ]. Furthermore,
extracts from Brazilian sponges have not been examined
for specific effects on the cell death-regulatory pathways .
Of particular interest is the marine sponge P. janeirensis.
To date, little informat ion is known about this specie, and
the only detected classes of compounds already described
in the reviewed literature for the genus Polymastia are
sterols and tetrahidroxiamide [48, 49]. Moreover, only two
works reported biological properties from P. janeirensis.In
an interesting work, Monks et al. (2002) described a
cytotoxic effect with organic extracts against three human
tumor cell lines (HT29, U373 and NCI- H460) with IC
50
concentrations ranging from 50 to 100 μ g/ml, as deter-
Invest New Drugs
26
mined by sulforhodamine assay. Moreover, aqueous ex-
tracts significantly retarded the migration of polymorpho-
nuclear leukocytes in a chemotactic assay [20]. However,
the mode of action underlying these biological activities
remains unclear. In the other work, an in vitro antiviral
activity was observed against the simian rotavirus RV-SA11
with aqueous extract of P. janeirensis, indicating that the
compounds found in the extract inhibited the late stage s of
rotavirus replication [50].
To our knowledge, this is the first report demonstrating
that marine sponge extracts of P. janeirensis induce cell
death and, at lower dose, induces caspase-dependent
apoptosis in U138MG glioma c ells. Furtherm ore, our
results suggested a selective cytotoxic effect on glioma cell
line compared to a normal cell culture, since the effect on
viability found in glioma cel ls was not observed in
astrocyte cultures with the lower dose (10 μg/ml). Besides,
the extracts herein tested were more effective than TMZ, a
potent inductor of apoptosis used for the treatment of
malignant gliomas. Thus, this marine sponge may be
considered as a good candidate for investigations and
development of news molecules, and further work to purify
and characterize the chemical structure(s) of the substance
(s) involved might yield new active compounds with
biological activities and potential in glioma cell treatment.
In this regard, bioassay-guided micro-fractionation by high
performance liquid chromatography with either photodiode
array or evaporative light-scattering detectors will be
performed. Once founded, the chemical structure of the
active const ituents will be established by usual spectro-
scopic analysis.
Acknowledgements This work was supported by FAPERGS,
CNPq, CAPES and PROPESQ-UFRGS. All experiments carried out
comply with current Brazilian laws.
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28
III. 2 - Artigo 2
Brazilian marine sponge Polymastia janeirensis induces oxidative cell death
through a caspase-9-apoptotic pathway in human U138MG glioma cell line
Artigo submetido para publicação no periódico Toxicology in Vitro
29
1
Brazilian marine sponge Polymastia janeirensis induces oxidative cell death
through a caspase-9-apoptotic pathway in human U138MG glioma cell line
Mario Luiz Conte da Frota Junior
a*
, Elizandra Braganhol
b
, Fabio Klamt
a
, Miriam
Anders Apel
c
, Beatriz Mothes
d
, Cléa Lerner
d
, Ana Maria Oliveira Battastini
b
, Amélia
Teresinha Henriques
c
, José Cláudio Fonseca Moreira
a
a
Centro de Estudos em Estresse Oxidativo (CEEO), ICBS - UFRGS, Porto Alegre,
Brazil;
b
Laboratório de Enzimologia, ICBS - UFRGS, Porto Alegre, Brazil;
c
Faculdade de Farmácia, UFRGS, Porto Alegre, Brazil;
d
Fundação Zoobotânica,
Museu de Ciências Naturais, Porto Alegre, Brazil;
Short running title: Effects of extracts from Polymastia janeirensis on glioma cells
*
Corresponding author: Mario Luiz Conte da Frota Jr, Departamento de Bioquímica,
ICBS - UFRGS. Rua Ramiro Barcelos 2600 - ANEXO, Porto Alegre, 90035-003, RS,
Brazil. Phone: +55 51 3308-5577; Fax: +55 51 3308-5535 (E-mail address:
mfrotaj[email protected]
).
* Manuscript
Click here to view linked References
30
2
Abstract
In this work we examine the apoptotic rote activated in response to treatments
with crude extracts (aqueous and organic) of
P. janeirensis. Yet, the formation of
intracellular ROS by glioma cells was measured. Exposure of cultures to both aqueous
and organic extracts (1-100 µg/ml) resulted in cell viability decrease, with an IC
50
12
µg/ml for both extracts. However, 10 µg/ml resulted in a higher incidence of cell death
that was almost entirely apoptotic, while a higher incidence of cell death by necrosis
was observed with doses
≥25 µg/ml. Moreover, inhibition of caspase-8 had no effect on
the amount of apoptosis induced by extracts, but inhibition of caspase-9 caused an
inhibition of apoptosis. We also observed a dose-dependent increase on ROS
production. However, 10 µg/ml is able to cause a milder oxidation that lead cells to die
by apoptosis, while higher doses of sponge extracts caused a stronger increase in ROS
production, leading cells to die by necrosis. This is a first report demonstrating that
marine sponge extracts of
P. janeirensis induces oxidative cell death through a caspase-
9 apoptotic pathway. Further work will concentrate on bioassay-guided micro-
fractionation to found the active constituents in the extracts.
Keywords: cancer; reactive oxygen species; apoptosis; marine sponges;
Polymastia
janeirensis
31
3
Introduction
Over the last years, marine organisms have been screened for a variety of
compounds with different biological activities. Among all organisms screened, sponges
represent one of the most promising sources of marine bioactive compounds particularly
for pharmaceutical leads (Proksch, 1994; Faulkner, 1998). Ecological factors, such as
competition for space with other sessile species, predation and symbiosis, have been the
most important causes for this large variety of secondary metabolites (Assman et al.,
2000).
In order to identify new active compounds with antitumor activity, extracts from
a number of species of marine sponges have been examined in various tumor cell lines,
as well as in animal models (Ahond et al., 1988; Burres et al., 1991; Fusetani et al.,
1992; Gunasekera et al., 1991; Kobayashi et al., 1989; Perry et al., 1988, 1990; Sakai et
al., 1986; ter Haar et al., 1996). At least two drugs (Ara-A and Ara-C) synthetically
derived from sponge metabolites have been clinically used in long-term cancer
treatments (Munro et al., 1994).
Of particular interest is the marine sponge
Polymastia janeirensis. Its intense
color and the apparent lack of predators suggest the presence of chemical defense
mechanisms. However, little information is known about this species. In an interesting
work, Monks et al. described a cytotoxic effect with organic extracts from
P. janeirensis
against three human tumor cell lines (HT29, U373 and NCI-H460). Yet, aqueous
extracts significantly retarded the migration of polymorphonuclear leukocytes in a
chemotactic assay (Monks et al., 2002). In other work, an
in vitro antiviral activity was
observed against the simian rotavirus RV-SA11 with aqueous extract of
P. janeirensis
(da Silva et al., 2006), and more recently, we reported that aqueous and organic extracts
32
4
of this marine sponge induces apoptotic cell death in human U138MG glioma cell line,
but not in a normal cell culture (da Frota et al., 2008). However, the precise apoptotic
pathway (receptor-dependent or receptor-independent) activated in response to
treatments remains unclear.
In this study, we examine the apoptotic rote activated in response to treatments
with crude extracts (aqueous and organic) of the marine sponge
P. janeirensis.
Moreover, in order to study if production of reactive oxygen species (ROS) was induced
by extracts, the formation of intracellular ROS by U138MG cells was measured. This
study is part of a collaborative program among several Brazilian institutions (Centro de
Estudos em Estresse Oxidativo, Universidade Federal do Rio Grande do Sul; Fundação
Zoobotânica do Rio Grande do Sul, Museu de Ciências Naturais; Departamento de
Bioquímica, Universidade Federal do Rio Grande do Sul; and Faculdade de Farmácia,
Universidade Federal do Rio Grande do Sul) for the collection and screening of
Brazilian marine sponges for biological activities, with the aim of identifying new
sponges species and novel molecules with promising and potentially useful therapeutic
activities.
Materials and methods
Sponge sampling, identification and extract preparation
Sponges samples were collected manually from exposed and semi-exposed
habitats, at depths of between 0.5 and 20 m, from locations on the coastline of the Estate
of Santa Catarina (southern Brazil). Taxonomic designation was based on scanning
electron microscope studies and on skeletal slides and dissociated spicule mounts.
33
5
Specimens of all materials are deposited in the Museu de Ciências Naturais-Porifera
(MCNPOR) collection of the Fundação Zoobotânica do Rio Grande do Sul, Brazil.
Aqueous and organic extracts were obtained as previously described (Monks et al.,
2002).
Cell culture
U138MG human glioblastoma cell line was obtained from the American Type
Culture Collection (Rockville, Maryland, USA). Cells were grown in culture flasks in
Dulbecco’s modified Eagle’s medium (DMEM)/15% fetal bovine serum (FBS) (v/v)
(Cultilab, Campinas, SP, Brazil) and seeded in 24-well plates (TTP plates) at densities
of 1 X 10
4
cells/well in 500 µl medium per well. Culture cells were maintained in 5%
CO
2
/95% air at 37
o
C and allowed to grow to confluence.
Treatments
Immediately before experiments, both the aqueous and organic extracts were
dissolved in water and DMSO, respectively, at a concentration of 1 mg/ml (w/v). The
amount of DMSO (maximum 0.25%) was proven not to affect the experiments. The
final concentrations of the extracts tested ranged from 1 to 100
g/ml. After reaching
subconfluence, the cultures were exposed to sponge extracts for 24 h. Control cultures
were performed with DMSO (0.25% final concentration) in the absence of extracts. In
experiments using permeable caspase inhibitors (Z-VAD-fmk, Z-IETD-fmk, Z-
LEDHfmk) (B & D Systems), cells were pre-incubated for 1 h and then treated with
34
6
extracts. None of the inhibitors used in this study had any effect on U138MG cell
viability at the concentrations used.
Assessment of glioma cell viability
Following treatments, cell viability was assessed by the MTT assay. This
method is based on the ability of viable cells to reduce 3-(4,5- dimethyl)-2,5 diphenyl
tetrazolium bromide (MTT) and form a blue formazan product. MTT solution (sterile
stock solution of 5 mg/ml) (Sigma) was added to the incubation medium in the wells at
a final concentration of 0.5 mg/ml. The cells were left for 60 min at 37
o
C in a
humidified 5% CO
2
atmosphere. The medium was then removed and plates were shaken
with DMSO for 30 min. The optical density of each well was measured at 550nm (test)
and 690nm (reference). Results were expressed as the percentage of cell viability
against the control.
Flow cytometry analysis
For annexin V/propidium iodide (AV/PI) staining, treated glioma cells (1x10
6
)
were washed twice in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na
2
HPO
4
.2H
2
O, 1.4
mM KH
2
PO
4
, pH 7.4) containing 1 mM EDTA (PBS-EDTA) and subsequently
trypsinized with 0.13 g/L trypsin in PBS-EDTA. Trypsin was inhibited with bovine
foetal serum 10%, and medium washes, and cells were combined and centrifuged (5
min, 200 x g, 4 °C). Cells were allowed to recover from trypsinization in complete
medium (30 min, 37 °C). Externalized phosphatidylserine (PS) was labeled (15 min, 0
°C) with 5 µl FITC-conjugated annexin V in 80 µl binding buffer (10 mM HEPES pH
35
7
7.4, 145 mM NaCl, 5 mM KCl, 1.0 mM MgCl
2
.6H
2
O, 1.8 mM CaCl
2
.2H
2
O). Propidium
iodide (PI) 2 µM was added 10 min prior to analysis on a FACScalibur flow cytometer
(BD PharMingen). When green fluorescence (FITC) was plotted against red
fluorescence (PI), distinct cell populations could be detected: viable cells (FITC
−/PI−),
apoptotic cells (FITC+/ PI− and FITC+/PI+), and necrotic cells (FITC−/ PI+), as
previously described (24). Ten thousand cells were analyzed per sample, and data were
reported as the percentage of apoptotic cells and necrotic cells.
Determination of intracellular ROS
Intracellular ROS were detected using an oxidation sensitive fluorescent probe,
2
′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), as previously described (LeBel
et al., 1992). Briefly, after treatments with extracts, cells were incubated with DCFH-
DA 10 μM for 30 min at 37
o
C. The DCFH-DA was first deacetylated by cellular
esterases to DCFH, which was converted to a green fluorescent product DCF by
intracellular reactive oxygen species produced by treated U138MG glioma cells. The
medium was discharged and cells were collected, resuspended in ice-cold PBS and
sonicated. The DCFH-DA oxidation was quantified from the fluorescence emission
intensity with an emission wavelength set at 535 nm and an excitation wavelength set at
485 nm.
Statistical analysis
Results were expressed as the mean ± SEM of at least three independent
experiments. Data were analyzed by a one-way analysis of variance (ANOVA), using a
36
8
Newman Keuls test to compare mean values across groups. When appropriate,
Student’s t-test was performed. Differences were considered to be significant when p
<0.05. Dose response curves were plotted, and the IC
50
values (concentrations at which
cellular effects are inhibited by 50%) were calculated using non-linear regression
analysis.
Results
Marine sponge extracts decreased U138MG glioma cell viability
In order to investigate the effect of marine sponge extracts on viability of
U138MG glioma cell line, cell cultures were treated with extracts and a MTT assay was
performed. Marine sponge extracts (1 and 5 µg/ml) did not decrease cell viability in
glioma cells (Fig. 1). However, exposure of glioma cell line to doses higher than 5
µg/ml resulted in a reduction of glioma cell viability, as evidenced by a diminished
ability of glioma cells to reduce MTT, with an IC
50
12 µg/ml for both aqueous and
organic extracts. Moreover, we observed that extracts from
P. janeirensis at doses
higher than 10 µg/ml have stronger cytotoxic effects, decreasing more than 90% of
glioma cell viability. The reduction in MTT staining suggests not only glioma cell
damage, but also a decrease in cell proliferation when compared with the control.
Cell death induced by marine sponge extracts
To determine whether the suppression of glioma cell proliferation was due to the
induction of necrosis or apoptosis, glioma cells were treated for 24 h and, at the end of
37
9
treatment, Annexin V-FITC-bound phosphatidylserine and red fluorescence of DNA-
bound PI in individual cells were measured using a flow cytometer as described on
“Materials and Methods”. As shown in Fig. 2A, marine sponge extracts at lower doses
(1 and 5 µg/ml) did not induce cell death in human U138MG glioma cell line, but
exposure of cultures to doses higher than 5 µg/ml resulted in cell death. However, 10
µg/ml of both extracts resulted in a higher incidence of apoptotic cell death, while a
higher incidence of cell death by necrosis was observed with doses
≥25 µg/ml. Since
higher doses have stronger cytotoxic effects and induced necrosis, we decided to
investigate only the effects of marine sponge extracts at 10 µg/ml.
To determine whether marine sponge extracts from
P. janeirensis activates the
extrinsic or intrinsic pathways of apoptosis, human U138MG glioma cells pre-incubated
with caspase-inhibitors and exposed to treatments for 24 h. As previously described,
cell death was prevented by the pan-caspase inhibitor Z-VAD (Fig. 2B), suggesting that
marine sponge extracts, at 10 µg/ml, induces caspase-dependent apoptosis in glioma
cells. Moreover, to determine which initiator caspase was activated in response to
extracts, cells were pre-incubated with either the cell-permeable caspase-9 inhibitor Z-
LEHD or the caspase-8 inhibitor Z-IETD for 1 h. We found that inhibition of caspase-8
had no effect on the amount of apoptosis induced by both aqueous and organic extracts.
In contrast, inhibition of caspase-9 caused an inhibition of apoptosis.
ROS production induced by marine sponge extracts
In order to investigate the effect of marine sponge extracts on intracellular ROS
production, glioma cells were treated for 24 h and a DCFH-DA assay was performed.
As shown in Fig. 3, extracts at lower doses (1 and 5 µg/ml) did not have any effect.
38
10
However, exposure of cell cultures to doses higher than 5 µg/ml resulted in a dose-
dependent ROS production. Similar to effects observed on viability of glioma cells, as
well as in the cell death, higher doses also have stronger effects.
Discussion
Apoptosis, or programmed cell death, is characterized by a number of well-
defined features (Hengartner, 2000). In contrast to necrosis, apoptotic cell death is
thought to be physiologically advantageous (Shacter et al., 2000; Anderson et al., 2002;
Anderson et al., 2003; Fadok and Henson, 2003; Lauber et al., 2004). In this way, we
have been previously reported that Brazilian marine sponge
P. janeirensis induces
apoptotic cell death in glioma cells, but not in a normal cell culture. Moreover, extracts
from this marine sponge were more effective than temozolomide, a potent inductor of
apoptosis used for the treatment of malignant gliomas. However, we have not described
the precise apoptotic pathway (receptor-dependent or receptor-independent) activated in
response to treatments. In this work, we examine the apoptotic rote activated in
response to marine sponge extracts. Moreover, the effect of extracts on intracellular
ROS production also was examined. The results will be discussed further below.
Apoptotic cell death occurs through at least two overlapping pathways referred
to as extrinsic and intrinsic. The extrinsic pathway is activated through ligand binding to
death receptors (members of the tumor necrosis factor receptor superfamily) at the cell
surface and activation of initiator caspase-8. In the intrinsic pathway, mitochondria play
a pivotal early role by releasing cell death signals into the cytosol and activating
caspase-9 (Fulda et al., 2001). Both pathways converge and activate executioner
39
11
caspase-3, which then cleaves intracellular protein substrates and causes cell death
(Korsmeyer et al., 2000).
Our results showed that marine sponge extracts, at doses higher than 5 µg/ml,
reduced the cell viability of glioma cultures. Nevertheless, we observed that higher
doses were stronger cytotoxic, decreasing more than 90% of cell viability. Furthermore,
higher doses caused cell death by necrosis, while a high percentage of apoptotic glioma
cells were counted with 10 µg/ml. For this reason, we decided investigate only the
effects of marine sponge extracts at 10 µg/ml in the apoptotic cell death. To determine
whether marine sponge extracts from
P. janeirensis activates the extrinsic or intrinsic
pathways of apoptosis, human U138MG glioma cells were pre-treated with caspase
inhibitors for 1 h and exposed to treatments for 24 h. We found that cell death was
prevented by the pan-caspase inhibitor Z-VAD, as previously described for us.
Moreover, we found that inhibition of caspase-8 with Z-IETD (a caspase-8 inhibitor)
had no effect on the amount of apoptosis induced by 10 µg/ml, but inhibition of
caspase-9 with Z-LEHD (a caspase-9 inhibitor) caused an inhibition of apoptosis. This
result suggests that extracts from Brazilian marine sponge
P. janeirensis cause
apoptosis through a receptor-independent pathway under our assay conditions.
It is well recognized that free radicals can regulate cell death, but the
mechanisms whereby oxidants activate the apoptotic machinery are not well
understood. Moreover, the mode of death (i.e. necrotic or apoptotic) depends on the
type, concentration, source and environment of the oxidants (Englert and Schacter,
2002). In normal conditions, there is a steady-state balance between the production of
ROS and their destruction by the cellular antioxidant system. When cells are exposed to
an oxidative stress various defense mechanisms are induced, including the antioxidant
enzymes superoxide dismutase (SOD) and catalase (CAT) (Halliwell and Gutteridge,
40
12
1999). In a previous work, we have demonstrated that there is a different modulation of
oxidative stress when compared radioresistant and radiosensitive glioma cell lines, and
these seem to be important for cell viability (Dal-Pizzol et al., 2003).
Some studies with marine natural products have reported a correlation between
free radicals and apoptosis in cancer cell lines (Koulman et al., 1996; Liu et al., 2005;
Liu et al., 2006). However, the mechanisms underlying these properties are still not well
understood. In this work, we observed a dose-dependent increase on ROS production at
doses higher than 5 µg/ml. Furthermore, similar to effects of marine sponge extracts on
viability of cell cultures, as well as in cell death, higher doses have stronger effects on
ROS production. The effect in cell viability and the strong effect in cell death observed
at higher doses are most likely through the high increase of ROS production (
100%
when compared to control) and a concomitant loss of membrane integrity, which is an
indication of cell death by necrosis. However, 10 µg/ml is able to cause a milder
oxidation that lead cells to die by apoptosis. The flow cytometry necrosis/apoptosis
measurement is consistent with this consideration.
Marine natural products have been prominently featured in the area of cancer
research. It is interesting to not that out of 18 preclinical investigating anticancer agents
derived from marine sources, six are from sponges (Nuijen et al., 2000; Newman et al.,
2000; Schwartsmann, 2000), implying that marine sponges are potential resources for
new anti-cancer agents. Although Brazil has the second most extensive coastline after
Australia, there are just few reports in which the authors have screened Brazilian sponge
extracts for biological activities. To date, only limited screening evaluations of extracts
of Brazilian marine sponges have been reported (Muricy et al., 1993; Monks et al.,
2002; Prado et al., 2004; Rangel et al., 2001). Furthermore, extracts from Brazilian
41
13
sponges have not been examined for specific effects on the cell death-regulatory
pathways and ROS production.
Of particular interest is the Brazilian marine sponge
P. janeirensis. However,
little information is known about this species, and the only detected classes of
compounds already described for the genus
Polymastia are sterols and
tetrahidroxiamide (Faulkner, 2001; Santafe et al., 2002). These compounds exhibited
significant cytotoxic activity toward human lung carcinoma (A-549), human colon
carcinomas (HT-29 and H-116), mice endothelial (MS-1), and human prostate
carcinoma (PC-3) cell lines in the range 0.5-10 µg/ml, but the mechanisms underlying
these properties were not reported. Related compounds belonging to these groups of
metabolites may be present in the tested extracts and/or could be even responsible for
the antiproliferative activity described in this work. However, further studies are
necessary to confirm this hypothesis.
To our knowledge, this is a first report demonstrating that marine sponge
extracts of
P. janeirensis induces oxidative cell death by apoptosis through a receptor-
independent pathway. Moreover, the biphasic effect (apoptosis or necrosis) observed in
response to different doses of marine sponge extracts may be related to the dose-
dependent increase on ROS production, since excess levels of free radicals can cause
oxidative stress, with an excessive activation of poly-ADP-ribose polymerase (PARP),
and a concomitant depletion of the ATP levels in the cells, leading cells to die by
necrosis. In agreement with this, some works have shown that oxidant treatment leads to
increased ATP consumption and subsequent cellular ATP depletion (Filipovic et al.,
1999; Schraufstatter et al., 1986). Additional studies are required to understand the
exact mechanism by which sponge extracts works to regulate ROS production and cell
death in glioma cells, as well as its significance in neoplasic transformation of normal
42
14
and previously injured cells. Moreover, further work will concentrate on bioassay-
guided micro-fractionation to found the active constituents in the extracts.
Conflict of interest statement - The authors declare that there are no conflicts of
interest.
Acknowledgements - This work was supported by FAPERGS, CNPq, CAPES and
PROPESQ-UFRGS. All experiments carried out comply with current Brazilian laws.
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Figure legends
49
21
Figure 1. Effects of marine sponge extracts on U138MG glioma cell viability. Cell
cultures were exposed to extracts for 24 h and the cell viability was assessed by the
MTT assay as described in “Material and methods”. Glioma cultures treated with
DMSO (control) was taken as 100% of cell viability. Data are the means ± S.E.M for
three individual experiments, and results were expressed as the percentage of cell
viability against the control.
∗
Statistically different from control, p < 0.05 (one-way
ANOVA).
Figure 2. Effects of marine sponge extracts on cell death. U138MG glioma cells were
treated for 24 h with different concentrations of sponge extracts
(A) or pre-treated for 1
h with caspase inhibitors and then exposed to sponge extracts
(B). Annexin V-FITC-
bound phosphatidylserine and red fluorescence of DNA-bound PI in individual glioma
cells were measured using a flow cytometer as described in “Materials and Methods”.
Legend: AE, aqueous extract; OE, organic extracts; Z-VAD, pan-caspase inhibitor (100
µM); Z-LEDH, caspase-9 inhibitor (100 µM); Z-IETD, caspase-8 inhibitor (100 µM).
The data represent averages from at least three independent experiments carried out in
triplicate.
Figure 3. Effects of marine sponge extracts on ROS production. Cells were exposed to
extracts for 24 h and intracellular ROS production was assessed by the DCFH-DA assay
as described in “Material and methods”. Glioma cells treated with DMSO (control) was
taken as 100% of ROS production. Data are the means ± S.E.M for three individual
experiments, and results were expressed as the percentage of ROS.
∗
Statistically
different from control, p < 0.05 (one-way ANOVA).
50
Figure 1
0
20
40
60
80
100
120
Control 1 µg/ml 5 µg/ml 10 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml
Percentage cell viability
Aqueous extracts Organic extracts
*
*
*
*
Figure 1
51
Figure 2
A)
0
10
20
30
40
50
60
70
80
90
100
cell death (%)
Control
AE
1
µg/ml
OE 1 µg/
ml
A
E
5
µg/
ml
O
E
5 µg/m
l
AE 10 µg/ml
OE 10 µ
g
/ml
AE 25 µg/ml
OE
25
µ
g/
ml
AE 5
0
µ
g
/
ml
OE 50
µ
g
/
m
l
A
E
1
0
0
µ
g
/
ml
OE
1
0
0 µ
g
/
ml
Necrosis
Apoptosis
B)
0
5
10
15
20
25
30
35
40
45
50
cell death (%)
C
o
ntr
o
l
A
E 10 µg/ml
O
E
1
0 µg/ml
Z-VAD + AE
Z-
V
A
D
+ OE
Z
-LEDH +
AE
Z-LE
D
H + OE
Z-I
ETD
+
A
E
Z-
IETD + OE
Necrosis
Apoptosis
Figure 2
52
Figure 3
0
50
100
150
200
250
Control
1
µ
g
/ml
5
µg
/
m
l
1
0
µ
g
/ml
25 µ
g
/ml
50 µg/ml
1
0
0
µ
g
/ml
Percentage ROS production
Aqueous extracts Organic extracts
*
*
*
*
Figure 3
53
III. 3 - Artigo 3
Anti-proliferative activities of extracts and isolated compounds from Polymastia
janeirensis
Artigo submetido para publicação no periódico The Biological Bulletin
54
Anti-proliferative activities of extracts and isolated compounds from Polymastia
janeirensis
Mario Luiz Conte da Frota Junior
a*
, Elizandra Braganhol
b
, Laura Bauerman
c
, Andrés
Delgado-Canedo
d
, Ana Lúcia Aboy
c
, Miriam Anders Apel
c
, Beatriz Mothes
e
, Cléa
Lerner
e
, Fábio Klamt
a
, Ana Maria Oliveira Battastini
b
, Amélia Teresinha Henriques
c
,
José Cláudio Fonseca Moreira
a
a
Centro de Estudos em Estresse Oxidativo (CEEO), ICBS - UFRGS, Porto Alegre,
Brazil;
b
Laboratório de Enzimologia, ICBS - UFRGS, Porto Alegre, Brazil;
c
Faculdade de Farmácia, UFRGS, Porto Alegre, Brazil;
d
Laboratório de Cardiologia
Molecular e Celular - IC/FUC, Porto Alegre, Brazil;
e
Fundação Zoobotânica, Museu
de Ciências Naturais, Porto Alegre, Brazil;
* Corresponding author: Mario Luiz Conte da Frota Jr, Departamento de Bioquímica,
ICBS - UFRGS. Rua Ramiro Barcelos 2600 - ANEXO, Porto Alegre, 90035-003, RS,
Brazil. Phone: +55 51 3308-5577; Fax: +55 51 3308-5535 (E-mail address:
mfrotaj[email protected]
).
Manuscript
Click here to download Manuscript: Anti-proliferative activities of extracts and isolated compounds from Polymastia janeirensis.doc
55
Marine sponges have been prominently featured in the area of cancer research.
Here, we performed a bioassay-guided fractionation to found the active constituents in
the extracts of the marine sponge Polymastia janeirensis. A strong cytotoxic activity
was observed against the human glioma cell line U138MG with the active fraction
isolated by HPLC-PDA. Furthermore, a high percentage of apoptotic glioma cells were
observed after treatments. Our results suggest that this marine sponge may be
considered a good source for new antiglioma metabolites. The chemical structure of the
active fraction will be established by usual spectroscopic analysis in further works.
Marine sponges are considered one of the most promising sources of new active
compounds with anticancer activity, and extracts from a number of species have been
examined in various tumor cell lines, as well as in animal models (1-8). In particular,
we have previously reported that crude extracts from Brazilian marine sponge
Polymastia janeirensis induces apoptotic cell death in glioma cells, but not in a normal
cell culture (9). This is particularly important because malignant gliomas are
characterized by an intrinsic resistance to apoptosis (10). Moreover, a growing body of
evidence suggests that pro-apoptotic agents have a significant antiglioma effect in
preclinical models. In a continuation of our search for new anticancer leads from marine
sponges, a bioassay-guided fractionation was performed to found the active constituents
in the extracts of the marine sponge
P. janeirensis. This study is part of a collaborative
program among several Brazilian institutions for the collection and screening of
Brazilian marine sponges for biological activities, with the aim of identifying new
sponges species and novel molecules with promising and potentially useful therapeutic
activities.
Sponge samples were collected and taxonomic designation was performed as
previously described (11).
P. janeirensis materials were ground with water three times
56
for 30 min to yield an aqueous extract (AE), and 1.002 g was subsequently extracted
with 1:1 CH
2
Cl
2
/MeOH (4 x 20 mL). The extract was filtered, the soluble fraction (SF1)
was evaporated under reduced pressure yielding 0.1468 g, and the insoluble fraction
(IF1) yielded 0.9319 g. The SF1 was found to be inactive and was not pursued. The
cytotoxic residue IF1 (0.006 g) was dissolved in MeOH (15 mL), the soluble fraction
(SF2) was dried under reduced pressure yielding 0.0008 g, and the insoluble fraction
(IF2) yielded 0.0049 g. The SF 2 was found to be inactive and was not pursued. The
cytotoxic residue IF2 was dissolved in water and subjected to high performance liquid
chromatography (HPLC) with a photodiode array (PDA) detector, and three regions
were collected (A1, A2 and A3).
Figure 1 shows the cytotoxic activities of different fractions from aqueous
extract of
P. janeirensis against human U138MG glioma cell line. The results obtained
revealed that IF2 have a strong cytotoxic effect, with an IC
50
= 2.5 µg/ml. Taking into
account the results obtained, the IF2 fractions was selected for isolation of the active
constituents. HPLC-PDA analyses are shown in Figure 2. Three regions in the
chromatogram, with retention time between 5, 25 and 35 min (A1, A2 and A3,
respectively), were collected and tested for their cytotoxic activities. As shown in Figure
3, the A3 fraction presented a strong anti-proliferative activity, with an IC
50
= 0.5
µg/ml. Moreover, the chromatographic profile suggests that the A3 is the active
constituent in the
P. janeirensis extracts, once only one component was observed in this
fraction.
Apoptotic cell death is thought to be physiologically advantageous than necrosis,
because the dying cells are cleared by phagocytosis prior to cell lysis and release of
inflammatory mediators (12-16). In this way, glioma cells were exposed to A3 at 0.5
µg/ml for 24 h and stained with YOPRO-1 and propidium iodide (PI). We observed a
57
high content of apoptotic cells in the treated cultures (Fig. 4), suggesting that A3
fraction induces apoptotic cell death in the U138MG glioma cell line. Further
investigations will concentrate on elucidation of the chemical structure of A3 by usual
spectroscopic analysis.
Acknowledgements - This work was supported by FAPERGS, CNPq, CAPES and
PROPESQ-UFRGS. All experiments carried out comply with current Brazilian laws.
Figure legends
Figure 1.
Effects of marine sponge fractions on glioma cell viability. U138MG human
glioblastoma cell line was obtained from the American Type Culture Collection
(Rockville, Maryland, USA). Cells were grown in culture flasks in Dulbecco’s modified
Eagle’s medium (DMEM)/15% fetal bovine serum (FBS) (v/v) (Cultilab, Campinas, SP,
Brazil) and seeded in 24-well plates (TTP plates) at densities of 1 X 10
4
cells/well in
500 µl medium per well. Culture cells were maintained in 5% CO
2
/95% air at 37
o
C and
allowed to grow to confluence. Immediately before experiments, the extracts were
dissolved in water. The cultures were exposed to treatments for 24 h. Control cultures
were performed with DMEM in the absence of extracts. Cell viability was assessed by
the MTT assay. MTT solution (sterile stock solution of 5 mg/ml) was added to the
incubation medium in the wells at a final concentration of 0.5 mg/ml. The cells were left
for 60 min at 37
o
C in a humidified 5% CO
2
atmosphere. The medium was then
removed and plates were shaken with DMSO for 30 min. The optical density of each
well was measured at 550nm (test) and 690nm (reference). Data are the means ± S.E.M
for three individual experiments, and results were expressed as the percentage of cell
58
viability against the control.
∗
Statistically different from control, p < 0.05 (one-way
ANOVA). Dose response curves were plotted, and the IC
50
values (concentrations at
which cellular effects are inhibited by 50%) were calculated using non-linear regression
analysis.
Figure 2. HPLC profile of IF2 extract. Chemical analyses were carried out with a 2695
Waters Alliance analytical chromatograph with a C
18
columm (3.5 µm - 4.6 x 75 mm;
Waters) and a PDA detector model 996 controlled by Empower Chromatograph
Software (Waters Corp., Milford, MA, USA). The chromatography separation was
carried out using a mobile phase, with water as solvent A and methanol as solvent B, at
a flow rate of 0.5 ml/min. The gradient program was as follows: 0-30% B (20 min), 30-
100% B (40 min), 100% B (50 min). The peaks were detected at 219 nm, and three
regions were collected (A1, A2 and A3).
Figure 3. Effects of isolated fractions from P. janeirensis by HPLC on U138MG
glioma cell viability. Cell cultures were exposed to A1, A2 and A3 aliquots for 24 h and
the cell viability was assessed by the MTT assay as described in the legend to Fig. 3.
Data are the means ± S.E.M for three individual experiments, and results were
expressed as the percentage of cell viability against the control.
∗
Statistically different
from control, p < 0.05 (one-way ANOVA). Dose response curves were plotted, and the
IC
50
values (concentrations at which cellular effects are inhibited by 50%) were
calculated using non-linear regression analysis.
Figure 4. Representative pictures of U138MG cell cultures stained with YOPRO-1 and
PI for apoptosis analysis. Briefly, PI stains necrotic cells, and YOPRO-1 incorporate
59
into DNA only in early apoptotic cells becoming highly fluorescent. At the end of the
treatment (24 h) with A3 fraction (0.5 µg/ml), glioma cells were incubated with
YOPRO-1 for (1 h, 0
o
C). PI (2 µM) was added 5 min prior to analysis on inverted
fluorescence microscope (Nikon Eclipse TE300; Nikon Inc., Melville, New York,
USA). Images were captured using a digital camera connected to the microscope.
References
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Laurent, C. Poupat, J. Pusset, M. Pusset, O. Thoisson, and P. Potier. 1988.
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Antitumor activity and biochemical effects of topsentin. Biochem Pharmacol 42:
745-751.
3. Fusetani, N., T. Sugawara, and S. Matsunaga. 1992. Theopederins A-E, potent
antitumor metabolites from a marine sponge
Theonella sp. J Org Chem 57: 3828-3832.
4. Gunasekera, S. P., M. Gunasekera, and P. McCarthy. 1991. Discodermide: a new
bioactive macrocyclic lactam from the marine sponge
Discodermia dissoluta. J Org
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Discorhabdin D, an antitumor alkaloid from the sponges Latrunculia brevis and Prianos
sp. J Org Chem 53: 4127-4128.
7. Sakai, R., T. Higa, C. W. Jefford, and G. Bernardinelli. 1986. Manzamine a, a
novel antitumor alkaloid from a sponge.
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Gunasekera, H. S. Rosenkranz, and B. W. Day. 1996.
Discodermolide, a cytotoxic
marine agent that stabilizes microtubules more potently than taxol.
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243-250.
9. da Frota, M. L. Jr., E. Braganhol, A. D. Canedo, F. Klamt, M. A. Apel, B.
Mothes, C. Lerner, A. M. Battastini, A. T. Henriques, and J. C. Moreira. 2008.
Brazilian marine sponge Polymastia janeirensis induces apoptotic cell death in human
U138MG glioma cell line, but not in a normal cell culture.
Invest New Drugs, in press.
10. Ziegler, D. S., A. L. Kung, and M. W. Kieran. 2008. Anti-apoptosis Mechanisms
in Malignant Gliomas.
J Clin Oncol 26(3): 493-500.
61
11. Monks, N. R., C. Lerner, A. T. Henriques, F. M. Farias, E. E. S. Schapoval, E.
S. Suycnaga, A. B. da Rocha, G. Schwartsman, and B. Mothes. 2002.
Anticancer,
antichemotactic and antimicrobial activities of marine sponges collected off the coast of
Santa Catarina, southern Brazil.
J Exp Mar Biol Ecol 281: 1-12.
12. Shacter, E., J. A. Williams, R. M. Hinson, S. Senturker, and Y. J. Lee. 2000.
Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma cell
apoptosis and phagocytosis.
Blood 96(1): 307-313.
13. Anderson, H. A., R. Englert, I. Gursel, and E. Shacter. 2002. Oxidative stress
inhibits the phagocytosis of apoptotic cells that have externalized phosphatidylserine.
Cell Death Differ 9(6): 616-625.
14. Anderson, H. A., C. A. Maylock, J. A. Williams, C. P. Paweletz, H. Shu, and E.
Shacter. 2003.
Serum-derived protein S binds to phosphatidylserine and stimulates the
phagocytosis of apoptotic cells.
Nat Immunol 4(1): 87-91.
15. Fadok, V. A., and P. M. Henson. 2003. Apoptosis: giving phosphatidylserine
recognition an assist--with a twist.
Curr Biol 13: 655-657.
16. Lauber, K., S. G. Blumenthal, M. Waibel, and S. Wesselborg. 2004. Clearance
of apoptotic cells: getting rid of the corpses.
Mol Cell 14: 277-287.
62
Figure 1
0
20
40
60
80
100
120
Control 0.1 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml 50 µg/ml 100 µg/ml
Percentage cell viability
AE
SF1
IF1
SF2
IF2
Figure 2
*
*
*
*
*
*
*
*
*
*
*
*
A1 A2 A3
63
Figure 3
0
20
40
60
80
100
120
Control 0.1 µg/ml 0.5 µg/ml 1 µg/ml 10 µg/ml
Percentage cell viability
A1
A2
A3
Figure 4
*
*
*
*
Control
A3
64
IV - DISCUSSÃO
65
IV - DISCUSSÃO
Cobrindo mais de 70% da superfície do planeta, os oceanos são uma das
principais fontes de novas moléculas com diferentes atividades biológicas e com
potencial aplicação no desenvolvimento de novos fármacos. Entre os diversos
organismos marinhos, as esponjas (Filo Porifera) representam a principal fonte de novos
metabólitos, muitas vezes inéditos na literatura e com estruturas químicas diferentes
daquelas encontradas em organismos terrestres. Isso se deve ao fato das esponjas
viverem fixas ao substrato. Como não podem se locomover, desenvolveram, ao longo
da evolução, uma série de mecanismos de defesas químicas, muitas vezes utilizadas
contra a ação de predadores, bem como na competição por espaço.
Quando comparadas a outros organismos marinhos, as esponjas apresentam a
maior taxa de compostos citotóxicos, com mais de 10% das espécies investigadas
apresentando alguma atividade biológica (Osinga et al., 1998; Zhang et al., 2003).
Estudos recentes, realizados pelo National Cancer Institute dos EUA, retratam as
esponjas marinhas como os organismos que mais produzem moléculas de alta
singularidade com possível interesse farmacológico e potencial utilização no tratamento
de doenças (Kelecom et al., 1991).
O Brasil possui a segunda maior extensão litorânea do mundo, ficando atrás
apenas da Austrália, sendo um dos países com o maior índice de biodiversidade do
mundo. Entretanto, poucos trabalhos explorando a diversidade química da nossa fauna
marinha foram realizados, uma vez que o principal foco da química de produtos naturais
no Brasil foi direcionado, por muitos anos, ao estudo de plantas medicinais. Isso mudou
somente na última década, e um considerável aumento no número de trabalhos
explorando as propriedades farmacológicas de esponjas coletadas no litoral brasileiro
66
pôde ser observado (Muricy et al., 1993; Rangel et al., 2001; Monks et al., 2002;
Berlinck et al., 2004).
Neste trabalho, um efeito anti-proliferativo foi observado quando a linhagem de
glioma humano U138MG foi exposta aos extratos (aquoso e orgânico) da esponja
marinha P. janeirensis (Fig. 1 e Fig. 2, Artigo 1). Contudo, uma diferença no padrão de
morte celular foi observada conforme a concentração de extrato utilizada. Nós
observamos que as doses mais altas (50 e 100 µg/ml) de ambos os extratos foram
extremamente citotóxicas, inibindo mais de 80% a proliferação e a viabilidade das
células. Além disso, uma morte celular por necrose foi observada com essas doses (Fig.
3, Artigo 1), enquanto uma morte celular por apoptose (Fig. 4, Artigo 1) foi observada
com a dose mais baixa (10 µg/ml).
Apoptose, ou morte celular programada, é caracterizada por uma série de
eventos bem definidos e acredita-se ser fisiologicamente mais vantajosa que a morte por
necrose. Por esse motivo, drogas que tenham como mecanismo de ação a indução de
morte celular por apoptose são de grande interesse. Surpreendentemente, uma nova
droga utilizada no tratamento de gliomas (Temozolomide) e que tem como principal
mecanismo de ação a indução de apoptose, foi menos efetiva que os extratos aqui
testados (Fig. 4, Artigo 1), nas nossas condições experimentais.
A morte celular programada pode ocorrer principalmente por duas rotas
referidas como extrínseca ou intrínseca. A primeira é ativada pela ligação de ligantes
nos chamados receptores de morte encontrados na superfície celular, com uma
conseqüente ativação da caspase-8. Já na rota intrínseca, a mitocôndria exerce um papel
fundamental, liberando sinais de morte para o citoplasma e ativando a caspase-9. Ambas
as rotas convergem e ativam a caspase-3, uma caspase executora que cliva proteínas
intracelulares e causa a morte celular.
67
Com o objetivo de elucidar a rota apoptótica ativada em resposta aos extratos,
inibidores de caspase foram utilizados. A morte celular foi prevenida quando as células
foram co-tratadas com Z-VAD, um inibidor geral de caspases. Além disso, quando as
células foram co-tratadas com Z-IETD, um inibidor específico de caspase-8, nenhuma
reversão no efeito anti-proliferativo foi observada. Entretanto, quando as células foram
co-tratadas com Z-LEHD, um inibidor específico de caspase-9, uma inibição da morte
apoptótica pôde ser observada (Fig. 2, Artigo 2). Esses resultados sugerem que os
extratos aqui testados induzem morte celular por apoptose pela via intrínseca.
Sabe-se que radicais livres podem regular o processo de morte celular, mas os
mecanismos envolvidos nessa regulação não são bem conhecidos. Além disso, o
mecanismo de morte celular (necrose ou apoptose) depende muito do tipo e da
concentração de radicais livres produzidos no ambiente celular (Englert e Schacter,
2002). Em um trabalho anterior do nosso grupo de pesquisa, foi demonstrada uma
importante diferença na atividade das principais enzimas antioxidantes de linhagens de
gliomas sensíveis ou resistentes à radiação, sugerindo um papel fundamental dos
radicais livres na viabilidade celular dos gliomas estudados (Dal-Pizzol et al. 2003).
Recentes estudos com produtos naturais de origem marinha têm demonstrado
uma correlação entre a geração de radicais livres e apoptose em diferentes linhagens de
câncer (Koulman et al., 1996; Liu et al., 2005; Liu et al., 2006). Ademais, foi
demonstrado recentemente que o composto geotidin A, um terpeno isolado da esponja
marinha Geodia japonica, induz a produção de radicais livres e induz morte por
apoptose dependente de caspase-3 na linhagem humana de leucemia promielocítica
HL60 (Liu et al., 2005). Contudo, a rota de morte ativada em resposta ao tratamento
com esse composto (dependente ou independente de receptor) não foi elucidada. Mais
tarde, o mesmo autor demonstrou que stelletin A, um outro composto isolado da mesma
68
esponja marinha, induz a produção de radicais livres e causa morte celular por apoptose,
com ativação da rota dependente de receptor.
Aqui, nós observamos um aumento de uma maneira dose-dependente na
produção de radicais livres quando as células foram expostas aos extratos aquosos e
orgânicos da esponja marinha P. janeirensis (Fig. 3, Artigo 2). Similar aos resultados de
viabilidade celular, as doses mais altas induziram um potente aumento na produção de
radicais livres (>100% comparado ao controle) e uma concomitante perda de
integridade da membrana celular, um indicativo de morte por necrose. Entretanto, a
dose de 10 µg/ml induziu um moderado aumento na geração de radicais livres, levando
a uma morte por apoptose (Fig. 3, Artigo 2). Esses resultados explicam, pelo menos em
parte, o efeito bifásico (necrose ou apoptose) observado. Níveis elevados de radicais
livres podem causar estresse oxidativo, com uma super ativação da PARP, e uma
conseqüente depleção dos níveis de ATP nas células, levando a uma morte por necrose.
Alguns trabalhos têm demonstrado que o tratamento com oxidantes e uma produção
exacerbada de radicais livres pode aumentar o consumo de ATP, causando uma
depleção do mesmo (Filipovic et al., 1999; Schraufstatter et al., 1986).
De acordo com a Organização Mundial de Saúde (OMS), a cada ano surgem
nove milhões de novos casos de câncer no mundo. No Brasil, segundo o Ministério da
Saúde, mais de quinhentos mil novos casos de câncer surgem por ano, e
aproximadamente 100 mil pessoas morrem anualmente (Instituto Nacional do Câncer,
2008). No entanto, apesar dos esforços para o desenvolvimento de novas drogas contra
o câncer, ainda não existem agentes realmente efetivos contra uma grande gama de
neoplasias. Por essa razão, é de extrema importância o desenvolvimento de novos
fármacos. Nesse sentido, extratos naturais continuam sendo a melhor fonte de novos
compostos com atividade anti-proliferativa, já que possuem uma enorme diversidade
69
química em suas constituições. É importante salientar que dentre 18 compostos de
origem marinha que estão sendo investigados pré-clinicamente contra diferentes tipos
de câncer, 6 são derivados de esponjas, ressaltando o enorme potencial desses animais
como fonte de novos metabólitos com propriedades anti-proliferativas.
Características particulares como fatores ambientais (exposição à alta
intensidade de luz e altas pressões de oxigênio) e uma geografia propícia (águas rasas)
fazem das esponjas do nosso litoral candidatas ideais para a obtenção de novos
fármacos, tornando-se relevante a pesquisa sobre a aplicação dos mesmos no tratamento
de doenças de alto impacto sócio-econômico. Sendo assim, a utilização de produtos
naturais extraídos de esponjas marinhas brasileiras pode ser considerada uma importante
ferramenta para o desenvolvimento de novos medicamentos, alvo principal da indústria
farmacêutica.
Os únicos compostos isolados a partir de esponjas do gênero Polymastia
encontrados na literatura são esteróis e tetrahidroxiamidas, obtidos da esponja P. tenax,
coletada na Colômbia. Esses compostos exibiram uma significante atividade citotóxica
contra linhagens de carcinoma humano de pulmão (A-549), carcinoma humano de cólon
(HT-29 e H-116) e carcinoma humano de próstata (Faulkner, 2001; Santafe et al.,
2002). Contudo, os mecanismos envolvidos no processo anti-proliferativo permanecem
desconhecidos. Além disso, somente dois trabalhos descrevem propriedades biológicas
em extratos de P. janeirensis coletadas no Brasil. No primeiro, foi observado um efeito
citotóxico em extratos orgânicos contra três linhagens de câncer (HT-29, U-373 e NCI-
H460), com um IC
50
entre 50 e 100 µg/ml. Ademais, extratos aquosos retardaram
significativamente a migração de leucócitos em um ensaio quimiotático (Monks et al.,
2002). Entretanto, o modo de ação dessas atividades biológicas permanece
desconhecido. Já no outro trabalho, uma importante atividade antiviral foi observada em
70
extrato aquoso contra o rotavírus RV-SA11 (da Silva et al., 2006). Nesse caso, o extrato
inibia os últimos estágios de replicação do rotavírus.
Com o objetivo de isolar da esponja marinha P. janeirensis os compostos
responsáveis pelas atividades observadas, um fracionamento bio-guiado foi realizado.
Diferentes frações foram obtidas após a extração com diferentes solventes, e a fração
mais ativa foi submetida à cromatografia líquida de alta eficiência com um detector de
arranjo de fotodiodos (PDA). Foram obtidas três frações, sendo que a fração A3 foi a
única fração ativa (Fig. 3, Artigo 3), com uma potente atividade apoptótica (Fig. 4,
Artigo 3). Além disso, o perfil cromatográfico obtido (Fig. 2, Artigo 3) sugere que a
fração A3 é o composto ativo que buscávamos, uma vez que somente um pico é
observado nessa região do cromatograma.
Nós também observamos uma diminuição no IC
50
ao longo do fracionamento.
Com o extrato aquoso bruto foi obtido um IC
50
= 15 µg/ml. Já com o isolado, um IC
50
=
0.5 µg/ml foi encontrado. Isso é extremamente importante quando a questão é o
desenvolvimento de um novo fármaco, uma vez que para a indústria farmacêutica não é
economicamente interessante quando a atividade desejada é encontrada apenas em
grandes concentrações.
Como falado anteriormente, drogas que tenham como mecanismo de ação a
indução de morte celular por apoptose são de grande interesse. Isso é particularmente
importante para gliomas, uma vez que eles são caracterizados por alterações em suas
rotas apoptóticas (Ziegler et al., 2008) e, mesmo com um tratamento agressivo
(radiação, cirurgia e quimioterapia), adquirem uma resistência intrínseca a esse
mecanismo de morte celular, dificultando ainda mais o tratamento. Nós demonstramos
aqui que o composto ativo isolado da esponja marinha P. janeirensis induz morte
71
celular por apoptose na linhagem de glioma humano U138MG, nas nossas condições
experimentais (Fig. 4, Artigo 3).
De uma maneira geral, o tratamento do câncer pode ser realizado através de
cirurgia, radioterapia e quimioterapia. De acordo com o estágio da doença, o sucesso do
tratamento geralmente é conseguido com a associação desses três tipos de tratamentos.
No entanto, problemas como uma baixa seletividade dos antineoplásicos e o
aparecimento de efeitos colaterais indesejados são muito freqüentes. Além disso, o
surgimento de resistência aos medicamentos conduz o tratamento para o uso combinado
de diferentes drogas e, conseqüentemente, aumenta a probabilidade de efeitos colaterais.
Os resultados aqui apresentados demonstram a existência de uma seletividade do
efeito citotóxico. Como pode ser observado na Figura 5, Artigo 1, os extratos testados
não induziram morte em culturas de astrócitos na dose em que foi observada morte por
apoptose nos gliomas. Apenas as concentrações que induziram necrose foram
citotóxicas para as células não transformadas. Isso é extremamente relevante quando
lembramos que diversos estudos já demonstraram uma resistência à terapia com
Temozolomide, o principal fármaco utilizado no tratamento de gliomas, e que tem como
um dos principais mecanismos de ação a indução de apoptose.
Esse é o primeiro trabalho demonstrando que extratos e frações isoladas da
esponja marinha P. janeirensis induzem morte celular, e que o mecanismo envolvido
está, pelo menos em parte, relacionado à produção de radicais livres. Ademais, uma
potente atividade apoptótica foi observada com o composto ativo isolado da esponja
marinha alvo desse estudo. Assim, baseados nos dados apresentados, nós propomos que
essa esponja marinha pode ser considerada uma boa candidata para futuras
investigações e para o desenvolvimento de novas drogas contra o câncer. É importante
salientar que o crescente estudo de propriedades farmacológicas em esponjas marinhas
72
em outros países contribuiu para a criação de programas de preservação desses animais,
bem como do eco-sistema em que eles vivem, e que a existência de programas de
pesquisa com esponjas nessas regiões reflete o maior número de moléculas e patentes
obtidas nesses países.
73
V - CONCLUSÃO
74
V. CONCLUSÃO
Os resultados obtidos nesta Tese apontam a esponja marinha Polymastia
janeirensis como uma importante fonte para o desenvolvimento de novos fármacos
contra o câncer. Neste trabalho, nós demonstramos que extratos e frações isoladas da
esponja marinha P. janeirensis induzem morte celular em uma linhagem de glioma
humano, e que o mecanismo envolvido está, pelo menos em parte, relacionado à
produção de radicais livres. Além disso, uma potente atividade apoptótica foi observada
com o composto ativo isolado da esponja marinha alvo desse estudo. Com esse trabalho,
esperamos contribuir de alguma maneira para o desenvolvimento de novas
metodologias para o tratamento dos gliomas. Por isso, ressalta-se a importância de
trabalhos como esse, uma vez que espécies podem ser extintas antes mesmo de serem
descritas e pesquisadas quanto às suas propriedades biológicas.
75
VI - PERSPECTIVAS
76
VI. PERSPECTIVAS
As principais perspectivas desse trabalho são 1) a elucidação estrutural do
composto ativo isolado da esponja marinha P. janeirensis por análises espectroscópicas
usuais, 2) desvendar quais os componentes da via intrínseca estão envolvidos na morte
celular programada observada em resposta aos tratamentos, 3) desvendar os
mecanismos pelos quais os radicais livres gerados pelos extratos induzem a morte
celular por necrose ou apoptose na linhagem estudada e 4) examinar o efeito
antitumoral do composto ativo isolado em outras linhagens tumorais.
77
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84
ANEXOS
- Carta de confirmação de envio do artigo científico “Brazilian marine sponge
Polymastia janeirensis induces oxidative cell death through a caspase-9-apoptotic
pathway in human U138MG glioma cell line” para publicação no periódico Toxicology
in Vitro;
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brazilian marine sponge Polymastia janeirensis” submetido para publicação no
periódico The Biological Bulletin, e carta de confirmação do envio.
85
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Title: Brazilian marine sponge Polymastia janeirensis induces oxidative cell death
through a caspase-9-apoptotic pathway in human U138MG glioma cell line
Authors: Mario Luiz Conte Frota Jr, Ph.D.; Elizandra Braganhol; Fabio Klamt; Miriam
Apel; Beatriz Mothes; Clea Lerner; Ana Battastini; Amelia Henriques; Jose Moreira
Article Type: Research Paper
Dear Dr. Mario Luiz Conte Frota Jr,
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Dear Mr. Frota Jr,
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87
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The Biological Bulletin
88
1
Condensed title: Bioactivities from marine sponge
Manuscript title: Antioxidant and procoagulant activities of extracts from Brazilian marine
sponge Polymastia janeirensis
Mario Luiz Conte da Frota Junior
a*
, Mariana Leivas Müller Hoff
a
, Luis Gustavo Ravazolo
a
,
Guilherme Antonio Behr
a
, Miriam Anders Apel
b
, Beatriz Mothes
c
, Cléa Lerner
c
, Amélia
Teresinha Henriques
b
, José Cláudio Fonseca Moreira
a
a
Centro de Estudos em Estresse Oxidativo (CEEO), ICBS – UFRGS, Porto Alegre, Brazil;
b
Faculdade de Farmácia, UFRGS, Porto Alegre, Brazil;
c
Fundação Zoobotânica, Museu de
Ciências Naturais, Porto Alegre, Brazil;
* Corresponding author: Mario Luiz Conte da Frota Jr, Departamento de Bioquímica, ICBS -
UFRGS. Rua Ramiro Barcelos 2600 - ANEXO, Porto Alegre, 90035-003, RS, Brazil. Phone: +55
51 3308-5577; Fax: +55 51 3308-5535 (E-mail address: [email protected]).
Keywords: Brazilian marine sponges; Biological activities; Reactive oxygen species; Natural
products.
Manuscript
Click here to download Manuscript: Antioxidant and procoagulant activities of extracts from Brazilian marine sponge Polymastia Janeirensis.doc
89
2
Abstract
This paper describes the antioxidant and procoagulant activities of extracts (aqueous and
organic) from Brazilian marine sponge Polymastia janeirensis. Moreover, the total phenolic
content of the extracts is evaluated. Both aqueous and organic extracts inhibited hydroxyl
production and prevented 2-deoxyribose degradation (IC
50
=163 g/ml and 129.5 g/ml,
respectively). The extracts also diminished nitric oxide production. Aqueous extracts were 2.3
times more effective than orgainc extracts in inhibiting this radical formation; the IC
50
of aqueous
extract was 150.95 g/ml and the IC
50
of organic extract was 345 g/ml. Yet, both aqueous and
organic extracts diminished the TBARS (an index of lipid peroxidation) content induced by
ABAP (IC
50
=186.4 g/ml and 158.9 g/ml, respectively). The determination of total phenolic
content showed that extracts presented an equally high content of phenolics, where phenolic
compounds constitue 6.79 ± 0.31% dry weight of aqueous extract and 6.90 ± 0.29% dry weight
of organic extract. In addition, extracts of P. janeirensis displayed a potent effect upon blood
coagulation, indicating a procoagulant activity. This is the first report demonstrating antioxidant
and procoagulant activities of extracts from Brazilian marine sponge P. janeirensis, suggesting
that this marine sponge should be considered as a good source of active compounds.
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3
Introduction
Marine organisms have provided a large proportion of the bioactive natural products
reported over the last 20 years. Among the many phyla found in the oceans, the best sources of
pharmacologically active compounds are sponges (Munro et al., 1999), with 10% of the
investigated species showing some biological activity. Ecological factors, such as competition for
space with other sessile species, predation and symbiosis, have been the most important causes
for this large variety of secondary metabolites (Assman et al., 2000).
Reactive oxygen species (ROS) and oxidative stress play an important role in the etiology
and progression of major human degenerative diseases. Moreover, oxidative stress affects
circulating proteins and is associated with an abnormal coagulative pattern (Shacter et al., 1995).
For this reason, there is a great interest in substances that act as endogenous and exogenous
antioxidants with potential application in biomedicine. In this context, analysis of marine
organisms chronically exposed to high levels of solar UV radiation often reveals active
antioxidants of unknown composition (Dunlap et al., 2003). This is particularly significant for
tropical marine organisms. During daylight exposure, the air saturation come across > 250%
(hyperoxic), which, in combination with high light intensities, can cause photooxidative toxicity
to the organisms via the photodynamic production of cytotoxic ROS (Dunlap et al., 1999).
Tropical marine sponges thus provide a rich resource for the discovery of novel products with
redox properties.
Although Brazil has the second most extensive coastline after Australia, there are just few
reports in which the authors have screened Brazilian sponge extracts for biological activities. To
date, only limited screening evaluations of extracts of Brazilian marine sponges have been
reported (Muricy et al., 1993; Monks et al., 2002; Prado et al., 2004; Rangel et al., 2001).
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4
Furthermore, extracts from Brazilian sponges have not been examined for specific effects on the
blood coagulation and free radicals production.
Of particular interest is the marine sponge Polymastia janeirensis Boury-Esnauls, 1973.
Its intense color and the apparent lack of predators suggest the presence of chemical defense
mechanisms. However, little information is known about this species, and the only detected class
of compounds already described for the genus Polymastia are sterols and tetrahidroxiamide
(Faulkner, 2001; Santafe et al., 2002). Moreover, only two works reported biological properties
from P. janeirensis (Monks et al. 2002; da Silva et al., 2006).
In this report, we investigate the in vitro effects of crude extracts from P. janeirensis
(aqueous and organic) collected from the Brazilian coastline on blood coagulation and their redox
properties. Moreover, the phenolic content of extracts was evaluated. This study is part of a
collaborative program among several Brazilian institutions (Centro de Estudos em Estresse
Oxidativo, Universidade Federal do Rio Grande do Sul; Fundação Zoobotânica do Rio Grande do
Sul, Museu de Ciências Naturais; and Faculdade de Farmácia, Universidade Federal do Rio
Grande do Sul) for the collection and screening of Brazilian marine sponges for biological
activities, with the aim of identifying new sponges species and novel molecules with promising
and potentially useful therapeutic activities.
Materials and methods
Reagents
All drugs were purchased from Sigma Chemicals (St. Louis, MO, USA).
Sponge sampling and identification
Sponge samples were collected manually from exposed and semi-exposed habitats, at
depths between 0.5 and 20 m, from locations on the coastline of Santa Catarina (southern Brazil).
Taxonomic designation was based on scanning electron microscope studies and on skeletal slides
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5
and dissociated spicule mounts. Specimens of all materials are deposited in the Museu de
Ciências Naturais–Porifera (MCNPOR) collection of the Fundação Zoobotânica do Rio Grande
do Sul, Brazil.
Extract preparation
Aqueous extracts were produced by the following procedure. Sponge materials were
ground together with sand and water three times for 30 min. The resulting extract (collected after
each 30 min) was subsequently filtered and freeze-dried. The remaining material was sequentially
extracted five times with a methanol/toluene mixture (3:1, v/v) by maceration over 5 days. The
resulting extract solution was then filtered and concentrated in a Rotavapor (organic extracts).
Immediately before experiments, both the aqueous and organic extracts were dissolved in water
and DMSO, respectively, at a concentration of 1 mg/ml (w/v). The amount of DMSO (maximum
0.25%) present was proven not to affect the experiments. The final concentrations of the extracts
tested ranged from 10 to 100 g/ml.
Hydroxyl scavenging activity
The formation of hydroxyl radicals (
OH) from Fenton reagents was quantified using 2-
deoxyribose oxidative degradation. The principle of the assay is the quantification of the 2-
deoxyribose degradation product, malondialdehyde, by its condensation with TBA (Hermes-
Lima et al., 1994). Briefly, typical reactions (control) were started by the addition of Fe(II) (6M
final concentration) to solutions containing 5 mM 2-deoxyribose, 100 M H
2
O
2
and 20 mM
phosphate buffer (pH 7.2). To measure extracts redox activity, different concentrations of extracts
(10 - 1000 g/ml) were added to the system before Fe (II) addition. Trolox (water-soluble
vitamin E analogue) and ascorbic acid were used as antioxidant standards. Reactions were carried
out for 15 min at room temperature and were stopped by the addition of 4% phosphoric acid (v/v)
93
6
followed by 1% TBA (w/v, in 50 mM NaOH). After boiling for 15 min, the absorbance of
solutions was measured at 532 nm. All tests were performed in triplicate. Percent values were
determined by comparing the absorbance of control (Fenton reagents) and test samples. Results
were expressed as IC
50
.
Scavenging activity of nitric oxide
Nitric oxide (NO) generated from sodium nitroprusside in aqueous solution at
physiological pH interacts with oxygen to produce nitrite ions, which were measured by the
Griess reaction (Green et al., 1982). The reaction mixture (3 ml) containing 10 mM sodium
nitroprusside in phosphate-buffered saline (control) and extracts at different concentrations (10 -
1000 μg/ml) were incubated at 37ºC for 60 min. Trolox and ascorbic acid were used as reference
antioxidant molecules. A 0.3 ml aliquot of the incubated sample was removed and 0.3 ml Griess
reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride in 2% H
3
PO
4
) was
added. The absorbance of the chromophore formed during diazotization of the nitrite with
sulfanilamide and subsequent coupling with naphthylethylene diamine was measured after 15
minutes at 540 nm. All tests were performed in triplicate. Percent values were measured by
comparing the absorbance of control (10 mM sodium nitroprusside in phosphate-buffered saline)
and test preparations. Results were expressed as IC
50
.
TBARS assay
The degree of lipid peroxidation was assayed by estimating the thiobarbituric acid
reactive substances (TBARS) by using the standard method with minor modifications (Draper
and Hadley, 1990). In brief, different concentrations of extracts (10 - 1000 g/ml) were added to
a solution rich in lipids - yolk of egg 1% (w/v) in buffer (Na
2
HPO
4
20 mM, NaH
2
PO
4
20 mM,
KCl 140 mM, pH 7,4). Trolox and ascorbic acid were used as antioxidant standards. Lipid
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7
peroxidation was initiated by adding 2,2’-azobis 2-metilpropionamidina (ABAP) 10 mM
(control). After 60 min at 37ºC, 0.3 ml of this reaction mixture was taken in a tube containing 0.6
ml of 15% TCA, centrifuged (10.000g, 10 min) and 0.5 ml from supernatant was separated and
mixed with 0.5 ml of 0.67% TBA (w/v). The mixture was heated in a hot water bath at 100 °C for
20 min to complete the reaction. The intensity of pink coloured complex formed was measured at
532 nm. Percent values were determined by comparing the absorbance of control (ABAP) and
test samples. Results were expressed as IC
50
.
Total phenolic content
The total phenolic content of the extracts was determined by an adapted colorimetric
assay in which tannic acid was used as standard (Singleton et al., 1999). Briefly, a 100 µl aliquot
of extracts was assayed with 100 µl of Folin reagent and 200 µl of sodium carbonate (35%, w/v).
The mixture was vortexed and diluted with distilled water to a final volume of 2 ml. After 10 min
the absorption at 725 nm was measured and the total phenolic content was expressed as tannic
acid equivalents (TAE µg/mg extract).
Clotting assay
A plasma coagulation assay adapted to the SpectraMax microplate ELISA reader was
used as described (Ribeiro et al., 1995). The procedure allows to follow clot formation and to use
kinetics parameters for the coagulation process. Briefly, 160 µl reactions containing 50 µl of
human citrated plasma were incubated for 5 min, with 80 µl of 20 mM HEPES, pH 7.4, with or
without varied amounts of extract (10, 25, 50 and 100 µg). Coagulation was triggered by adding
CaCl
2
to a final concentration of 10 mM, and clot formation was monitored at 37
o
C in the
SpectraMax system at 650 nm. To access calcium-independent procoagulant activity, EDTA was
added, in place of CaCl
2
, to a final concentration of 10 mM.
Statistical analysis
95
8
Results were expressed as the mean ± SEM of at least three independent experiments.
Data were analyzed by a one-way analysis of variance (ANOVA), using a Newman Keuls test to
compare mean values across groups. When appropriate, Student’s t-test was performed.
Differences were considered to be significant when P <0.05. Dose response curves were plotted,
and the IC
50
values (concentrations at which effects are inhibited by 50%) were calculated using
non-linear regression analysis.
Results
Hydroxyl scavenging activity
Hydroxyl radicals were formed in solution and were detected by their ability to degrade 2-
deoxyribose into fragments that formed a pink chromogen upon heating with TBA. When
extracts were added to the reaction mixture, they removed
OH and prevented sugar degradation.
The IC
50
of aqueous extract was 163 g/ml and the IC
50
of organic extract was 129.5 g/ml
(Table 1).
Scavenging activity of the nitric oxide
The results of the inhibitory effect of aqueous and organic extracts on NO production are
shown in Table 1. Aqueous extract was 2.3 times more effective in attenuating nitric oxide
production than organic extract. The IC
50
of aqueous extract was 150.95 g/ml and the IC
50
of
organic extract was 345 g/ml.
TBARS assay
Table 1 shows the antioxidant capacity of extracts and reference compounds in a
lipoperoxidative system. Both aqueous and organic extracts prevented lipid peroxidation induced
by ABAP at different concentrations, with an IC
50
of 186.4 g/ml for aqueous extract and 158.9
g/ml for organic extract.
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9
Total phenolic content
The determination of total phenolic content in aqueous and organic extracts of marine
sponge P. janeirensis showed that both extracts presented an equal content of phenolics, where
6.79 ± 0.31% dry weight of aqueous extract and 6.90 ± 0.29% dry weight of organic extract are
phenolic compounds (Fig. 1).
Blood coagulation
To analyze the effect of marine sponge extracts upon clotting, human citrated plasma was
incubated with extracts at different doses prior to the induction of coagulation with calcium. As
shown in fig.2, extracts of marine sponge P. janeirensis displays a potent effect upon blood
coagulation. While the control plasma (with no sample added) took over 4 min to initiate
coagulation after the addition of calcium, plasma incubated with aqueous extract of the P.
janeirensis took around 1.5 min to start coagulation (fig.2A), and 3 min with organic extract
(fig.2B), indicating procoagulant activity. To access calcium-independent procoagulant activity,
EDTA was added, in place of CaCla
2
, but any effect was observed (data not shown). Moreover,
normal plasma was not capable of clotting when neither extract nor calcium was added (data not
shown).
Discussion
Diverse biological processes are modulated by free radicals, and ROS are recognized as a
cause of immediate cellular injury leading to cell death or apoptosis. ROS can also lead to
progressive accumulation of biomolecular damage and, consequently, are involved in many
physiological (i.e. aging) and pathological (i.e. cancer) processes (Ames et al., 1993). Moreover,
oxidative stress may promote an abnormal coagulative pattern and alter the structure and the
function of coagulative proteins, as observed in several diseases (de Cristofaro et al., 2002;
Abraham, 2000).
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1
The hydroxyl (
OH) radical is probably the most potent ROS, and the probable initiator of
the chain reactions which form lipid peroxides and organic radicals. Peroxidation of
biomembrane lipids is known to cause serious damage to tissues and cells. This damage in the
cellular membrane in turn leads to an increase in calcium leakage from internal storage sites in
the cell. This will trigger an increase in nitric oxide synthesis via the activation of calmodulin-
dependent nitric oxide synthase (Lai and Singh, 2004). Nitric oxide can further amplify iron-
mediated free radical formation via its effects on iron metabolism and release of iron from ferritin
(Richardson and Ponka, 1997; Reif, 1990). Thus, the effects will be amplified. Natural products
that specifically target free radical production and blood coagulation are, therefore, promising for
experimental approaches to the treatment of various malignancies.
Our work suggests that extracts of marine sponge P. janeirensis have antioxidant activity.
In general, both aqueous and organic extracts decreased the
OH production, attenuated NO
generation and prevented lipid peroxidation. Nevertheless, the scavenging activity of the nitric
oxide was clearly greater in the aqueous extract (2.3 times more effective than organic extract).
Metabolites consisting of benzenoid and terpenoid parts are some of the most active
marine metabolites. However, the antioxidant activity of marine phenolic metabolites has been
poorly studied. From sponges, a few examples of antioxidants have been reported; terpenoid
phenols and sesquiterpenequinones isolated from Sarcotragus spinulosus have been shown to
possess antioxidant activity (Utkina et al., 2004).
It is know that phenolic compounds may act as antioxidants by scavenging reactive
oxygen and nitrogen species and chelating redox-active transition metal ions in vitro, however, it
has been demonstrated that the performance of these secondary metabolites in oxidative systems
depends on activity-structure relationships (Cao et al., 1997; Rice-Evans et al., 1996). In this
98
1
work, we used total phenolic content to estimate the contribution of these substances in sponge
extracts to the performance in different antioxidant assays. Although this method could be
overestimating total phenolics, it is so far the only single and widely used method for estimating
total phenols (Deepa et al., 2007). Our results demonstrated that total phenolic content of
aqueous extract was equal to organic extract. However, the differences observed in the
scavenging activity of the nitric oxide suggest qualitative differences in the phenolic composition
of the extracts, and this point needs further investigations.
In a recent work, da Silva et al. (2007) reported that the antioxidant activities of Bauhinia
microstachya extracts (a brazilian native plant widely used in folk medicine to treat various
ailments) are linked to their polyphenol content. Interestingly, our results demonstrated a high
content of phenolic compounds in both aqueous and organic extracts from P. janeirensis,
suggesting that extracts of the Brazilian marine sponge P. janeirensis should be considered as a
good source of natural antioxidants. In this context, there are some studies showing that the
phenolic content of an extract could be positively correlated to the antioxidant potential
(Hukkanen et al., 2006; Kuti and Konuru, 2004).
We also investigated the action of sponge extracts upon clotting. Our findings show, for
the first time, the presence of procoagulant activity in marine sponge extracts. However, the
mechanism by which this works is unknown, and studies are being performed in order to better
characterize the pathway (extrinsic or intrinsic) here activated.
Sponges are known to produce a wide variety of chemical compounds and chemical
analyses are the subject of an impressive body of literature. However, there are just few reports in
which the authors have screened Brazilian sponge extracts for biological activities. To our
knowledge, this is the first report demonstrating that marine sponge extracts from P. janeirensis
have antioxidant activities and affect blood coagulation in vitro . Moreover, our findings suggest
99
1
that this marine sponge should be considered a good source of natural antioxidants due to their
high phenolic content. Additional studies are required to understand the exact mechanism by
which these extracts works, and further work to purify and characterize the chemical structure(s)
of the substance(s) involved might yield new active compounds with biological activities and
potential application in biomedicine.
Acknowledgements - This work was supported by FAPERGS, CNPq, CAPES and PROPESQ-
UFRGS. All experiments carried out comply with current Brazilian laws.
100
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Table 1. Antioxidant potential of extracts from P. janeirensis in different in vitro systems
Sample test Scavenging activity against
hydroxyl radical in IC
50
Scavenging activity against
nitric oxide radical in IC
50
Inhibition of lipoperoxidation
in IC
50
Aqueous extract
Organic extract
Trolox
Ascorbic acid
163 ± 0.1 150.95 ± 0.35 186.4 ± 0.1
129.5 ± 0.2
345 ± 0.4
158.9 ± 0.1
1.15 ± 0.02
0.20 ± 0.01
13.6 ± 0.08
6.56 ± 0.01 N.D 4.1 ± 0.02
a) IC
50
: sample concentration required for 50% inhibition. Aqueous and organic extracts are µg/ml. References standards are
mM in hydroxyl and nitric oxide assay; in lipoperoxidation assay standards are µM. N.D: not detected. The results are expressed as
means ± SEM of at least three independent experiments.
106
1
Figure legends
Figure 1. Total phenolic content of aqueous and organic extracts from Polymastia janeirensis.
The results are expressed as tannic acid equivalents (TAE µg/mg extract). Bars represent the
means ± SEM of at least three independent experiments.
Figure 2. Effect of sponge extracts from Polymastia janeirensis upon coagulation of human
plasma. (A) Aqueous extracts; (B) Organic extracts. Fifty microliters of normal citrated human
plasma were incubated with 80 µl of 20 mM HEPES, pH 7.4, with or without extract (10, 25, 50
and 100 µg/ml). Coagulation was triggered by adding 10 µl of 100 mM CaCl
2
. In the control
reaction (■) no extract was added. In the experimental reactions, plasma was incubated with
either 10 (▲), 25 (), 50 () or 100 µg (●) of extract prior to the addition of calcium. (♦)
Plasma was incubated with extract, and 10 Mm EDTA was added instead of CaCl
2
. Reactions
(clot formation) were monitored in the SpectraMax system at 650 nm and 37
o
C. The data are
representative of 3 independent experiments carried out in triplicate.
107
Figure 1
0
10
20
30
40
50
60
70
80
90
100
Aqueous extract Organic extract
TAE (µg/mg)
Figure 1
108
Figure 2
0
0.05
0.1
0.15
0.2
0.25
0
1
2
3
4
5
6
7
8
9
10
11
12
1
3
1
4
1
5
time (min)
Abs
0
0.05
0.1
0.15
0.2
0.25
0
1
2
3
4
5
6
7
8
9
10
11
1
2
1
3
1
4
15
time (min)
Abs
A)
B)
Figure 2
109
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