6.4 Avaliação da Citotoxicidade em Células Tumorais
6.5.3 Avaliação da atividade citotóxica de CTL
A avaliação da atividade citotóxica de CTL em células tumorais utilizou o método do MTT. Este é um teste amplamente utilizado para determinação da viabilidade de células isoladas. No ensaio o MTT, um sal de tetrazólio solúvel em água, é convertido em cristais de formazan de cor púrpura, insolúveis em água, após clivagem do anel de tetrazólio por desidrogenases mitocondriais e outras enzimas lisossomais (LIU et al., 1997). Na verdade, o MTT não interage diretamente com as desidrogenases e sim com os seus subprodutos, NADH e NADPH (LIU et al., 1997). Os cristais de formazan são solubilizados, formando assim um composto colorido cuja medição da densidade óptica é feita em espectrofotômetro.
A intensidade do produto colorido formado é diretamente proporcional ao número de células viáveis presente na amostra, confirmando a capacidade redutora do sistema sobre o MTT (LIU et al., 1997; HEINRICH et al, 2005). Outros estudos têm examinado a correspondência entre os resultados obtidos durante contagem direta de células e confirmou-se que a análise com MTT é um indicador válido do número de células viáveis (KIM et al., 2009). Como resultado do ensaio, verificou-se que CTL não foi hábil em reduzir significativamente a viabilidade celular dos gliomas de (Figura 26).
Figura 25. Ensaio de citotoxidade de CTL em células tumorais.
Fonte: Elaborado pelo autor. Gráfico de “Concentração × Viabilidade celular” de CTL. Média ± Média do erro padrão (n = 3).
Estudos anteriores mostraram a capacidade de da lectina de Canavalia ensiformis (ConA) de induzir morte autofágica de células de glioma na linhagem de células de glioma humano U87. Foi mostrado que essa atividade se deve a regulação da proteína MT1-MMP (proteína de membrana – metaloproteinase tipo 1), uma glicoproteína fortemente expressa em
gliobastomas radio- e quimioresistentes. Essa proteína é responsável por mediar a sinalização pró-apoptótica e autofágica em células de câncer do cérebro. A lectina foi capaz de induzir a atividade da proteína com consequente aumento de vacúolos ácidos característicos de processos autofágicos (BELKAID et al., 2007; GINGER et al., 2000; PRATT; ROY; ANNABI, 2012).
Outros trabalhos também demonstraram que as lectinas de Canavalia ensiformis,
Canavalia brasiliensis e Cratylia floribunda foram citotóxicas, inibindo tanto a viabilidade
quanto a proliferação celular de diferentes linhagens tumorais murina (J774) e humanas (MCF- 7, HL-60 e MOLT-4) (FAHEINA-MARTINS, 2009; FAHEINA-MARTINS et al., 2012). Foi possível observar no trabalho que houve uma variação na ação das lectinas em cada linhagem, Sendo ConA, mais tóxica sobre as linhagens, pelo fato desta encontrar-se completamente tetramérica em pH fisiológico, conseguindo exercer um efeito mais pronunciado que ConBr e CFL, que se apresentam numa mistura de dímeros e tetrâmeros em pH fisiológico (SANS- APARÍCIO et al., 1997; CALVETE et al., 1999).
7 CONCLUSÃO
Os resultados apresentados neste trabalho de tese forneceram dados importantes sobre a caracterização estrutural e bioquímica da lectina de sementes da espécie pertencente a tribo Dalbergieae, Centrolobium tomentosum. A lectina demonstrou alta similaridade de estrutura primária com outras lectinas de sua tribo. Além disso, os resultados sobre sua sequência corroboraram a possibilidade da existência de dois grupos na tribo Dalbergieae, apresentando especificidade a carboidratos, sítios de glicosilações e, principalmente, processamentos pós-traducionais diferentes.
A partir de dados de cristalografia de raios X foram obtidas duas estruturas de CTL, uma de forma monoclínica e a outra tetragonal, sendo elucidados dados importantes sobre suas interações proteína-ligante. Esses foram aprofundados com o estudo de Docking molecular. A lectina xibiu forte afinidade para manosídeos, sendo essa característica demonstrada tanto por co-cristalização, como por Docking molecular.
De posse desses resultados, a lectina pode ser utilizada para reconhecer glicanos específicos envolvidos eventos biológicos, tanto para entendimento do evento, quanto para aplicação biotecnológica. Um bom exemplo disso foi a indução do efeito edematogênico demonstrado por CTL no modelo de edema da pata, mostrando uma resposta inflamatória aguda, com a participação de seu domínio de reconhecimento a carboidratos. A elevada afinidade por complexos de manose apresentada pela sua estrutura cristalográfica pode explicar os resultados obtidos nos testes em modelos de inflamação.
Este estudo apresentou, pela primeira vez, a estrutura tridimensional de uma lectina nativa, específica à manose, pertencente à tribo Dalbergieae.
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