In vitro interaction of the native lectin isolated from the green seaweed Caulerpa cupressoides var. lycopodium (Caulerpaceae, Bryopsidales) against cancer HL-60 cells / Interação in vitro da lectina nativa isolada da alga marinha verde Caulerpa cupressoides var. lycopodium (Caulerpaceae, Bryopsidales) contra células cancerígenas LH-60
DOI:
https://doi.org/10.2312/Actafish.2016.4.2.117-124Keywords:
green alga, agglutinin, cytotoxicity, diagnostic tool / alga verde, aglutinina, citotoxicidade, ferramenta diagnósticoAbstract
Abstract Seaweeds have structurally diverse metabolites with biotechnological importance, including lectins, considered a variable class of proteins which bind reversibly to specific carbohydrates. The Caulerpa cupressoides (Chlorophyta) lectin (CcL) exhibiting in vivo pharmacological actions has been described; however, CcL-based diagnostic tool for cancer has not been investigated. This study analyzed molecular weight, cytotoxicity and the in vitro interaction against human promyelocytic leukemia cell lines (HL-60). Extraction of CcL was performed in 25 mM Tris-HCl buffer (TB) (4 h stirring, pH 7.5) and then CcL was purified by ion-exchange chromatography on DEAE-cellulose column using TB containing 0.5 M NaCl, which eluted protein peak exhibiting haemagglutinating activity against trypsin-treated rabbit erythrocytes. Polyacrilamide gel electrophoresis in the presence of β-mercaptoethanol revealed to be a dimeric protein (17 kDa) by comparison with the markers trypsin inhibitor and α-lactoalbulmin. CcL did not cause cytotoxicity up to 500 μg mL-1 in HL-60 cells using MTT reduction assay; but, showed agglutination (125, 250 and 500 μg mL-1) in comparison to Concanavalin A (50 μg mL-1) based on photomicrographic analysis. The results allow us to consider CcL as a promising diagnostic tool for HL-60 cells in further studies.
Resumo As algas marinhas possuem metabólitos diversos estruturalmente com importância biotecnológica, incluindo lectinas, consideradas uma classe variável de proteínas as quais reversivelmente se ligam a carboidratos específicos. A lectina de Caulerpa cupressoides (Chlorophyta) (LCc) tem mostrado ações farmacológicas in vivo. Entretanto, não tem sido investigada como ferramenta diagnóstica para o câncer. Analisou-se o peso molecular, citotoxidade e a interação in vitro com células de leucemia promielocítica da linhagem (LH-60). A extração de LCc foi desenvolvida em tampão Tris-HCl 25 mM (TT) (4 h sob agitação; pH 7,5) e em seguida, a LCc foi purificada por cromatografia de troca iônica em coluna de DEAE-celulose usando TT contendo NaCl 0,5 M, da qual pico protéico eluído apresentou atividade hemaglutinante contra eritrócitos de coelho tratados com tripsina. A eletroforese em gel de poliacrilamida, na presença de β-mercaptoetanol, revelou uma proteína dimérica (17 kDa) por comparação com os marcadores inibidor de tripsina e α-lactoalbumina. A LCc não mostrou, em ensaio de redução pelo MTT, citotoxicidade até 500 μg mL-1 em células LH-60. Contudo, na análise fotomicrográfica, mostrou aglutinação (125, 250 e 500 μg mL-1) em comparação a Concanavalina A (50 μg mL-1). Os resultados permitem a considerar LCc como uma ferramenta diagnóstica promissora em estudos futuros realizados com células LH-60.
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