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INTRODUCTION AND OBJECTIVES: In vitro and clinical antitumor efficacy of Viscum album is mainly attributed to the aqueous preparations. However, Viscum album ethanolic extracts (VAE), as mother tinctures prepared from different host trees, have demonstrated antiproliferative activity in 2D and 3D cellular models. MATERIAL AND METHODS: Viscum album subspecies album, austriacum, and abietis were harvested from five different host trees to prepare their respective mother tinctures. Phytochemical analyses were performed by thin layer chromatography, high-performance liquid chromatography and liquid chromatography-high resolution mass spectrometry. The antitumor activity using 2D cell models was evaluated by WST-1 and Annexin V-7AAD in Yoshida, Molt-4 (tumor cell lines), and NIH/3T3 (non-tumor cells). The glycolytic pathway analysis in 2D model investigated cell death mechanism, MTT and crystal violet evaluated the effect of the mother tinctures in human breast adenocarcinoma (MDA-MB-231) 3D cell line model. RESULTS AND CONCLUSIONS: Phenolic acids, flavonoids and lignans were the main chemical classes identified in VAE. V. album ssp. abietis from Abies alba (VAA) was the most effective to induce 2D in vitro cellular effects. Necrotic damage, which was host tree-, time- and dose- dependent, was observed by colorimetric and FACS methodologies, with different selectivity to non-tumor and tumor cell lines. The MDA-MB-231 glycolytic pathway in the 2D model showed a decrease in glucose consumption and extracellular lactate production. PFK (6-phosphofructo-1-kinase) and PK (Pyruvate kinase) activities were inhibited by VAA and VAQ (V. album ssp. album growing on Quercus sp.) after 48h of incubation. Regarding to 3D cell models and seasonal aspects, VAA and VAQ from summer harvests induced higher cytotoxic damage than winter preparations, with 49% and 42% tumor viability reduction, respectively. VAE presented antitumor activity in both 2D and 3D models. The glycolytic pathway (PFK and PK enzymes) should be an important target and needs further investigation.
Christian Ferreira
Boa tarde, Michelle!
Parabéns pelo trabalho!
Verificando o resumo e o pôster, notei que não foi apresentado os resultados da PFK e da PK. Além disso, a figura 3 não possui informações sobre qual o teste que está sendo avaliado pela citometria de fluxo, e o que significa as barras pretas, brancas e cinzas.
Foi realizado o ensaio de MTT com as células MDA-MB-231 tratadas com a tintura no modelo 2D, antes de partir para o modelo 3D?
Na figura 4-A, o MTT demonstra que A-V e Q-V reduziram significativamente a viabilidade, porém na figura 4-B, é mostrado os resultados com A-I e P-I, que não reduziram a viabilidade.
Qual foi a sua participação efetiva na obtenção dos resultados? E quais são os próximos passos?
Danielly Ferraz da Costa
Prezada Michelle,
parabéns pelo trabalho! Gostaria de saber por qual método vocês desenvolvem a cultura tridimensional de MDA-MB-231 (considerando que essa é uma linhagem que não costuma formar espontâneamente esferoides bem delimitados como os das imagens). Além disso, as atividades enzimáticas (PFK e PK) só foram medidas após 48h de exposição? Qual a explicação para que o efeito tenha sido observado após esse período e não mais precocemente?
Obrigada!
Atenciosamente,
Danielly Ferraz.
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