5-FLUOROURACIL CHRONIC EXPOSURE INDUCES MORPHOLOGICAL FEATURES EMT-RELATED IN COLORECTAL CANCER CELLS.
Details
- Presentation type: IC - Undergraduate Students
- Track: 2. Cellular Biology
- Keywords: 5-fluorouracil; Colorectal cancer; Chemoresistance; EMT; Morphological features;
- 1 Instituto Nacional de Câncer
- 2 Instituto Nacional de Câncer - INCA
- 3 INSTITUTO NACIONAL DO CANCER
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Abstract
INTRODUCTION AND OBJECTIVES: Colorectal cancer (CRC) is the third most common cancer in the world. Over the last decades, 5-fluorouracil (5-FU) remains one of the most effective used drugs to treat CRC. Despite several advantages, chemoresistance is a critical limitation to the clinical application of 5-FU. Growing body of evidence suggests that epithelial-mesenchymal transition (EMT) is associated with acquired resistance to cancer therapy. EMT is a complex process that regulates changes in cell morphology and mechanical properties of the cells. Modulation of elastic properties of cells during transition allows the cells to detach from their neighbors and the underlying basement membrane to facilitate their easy migration and invasion during metastasis. Therefore, the elucidation of the profile of the chemoresistance cells is of great importance to obtain improved treatment outcomes. The aim of this study was to characterize the morphological features of chemoresistance CRC cells. MATERIAL AND METHODS: The human colorectal cancer cell line HCT-116 were exposed to increasing concentrations of 5-FU over a period of 12-24 months to obtain resistant cells. Cell viability and IC50 were evaluated by MTT assay. Morphometric parameters of resistant cells such as circularity, perimeter and cell area were analyzed and compared with parental cells by phase-contrast microscopy at a magnification of 20x and measured using ImageJ software. The circularity was calculated using the formula 4π×[Area] / [Perimeter]. At least 200 highly distinct cells were averaged for each condition. Statistical analyses were performed using the GraphPad Prism program. RESULTS AND CONCLUSIONS: We initially showed that the cell viability of HCT-116 5-FU resistant cells (HCT-116 5-FUR) was higher than HCT-116 parental cells (HCT-116P), and IC50 values of HCT-116 5-FUR was approximately 300 times higher than HCT-116P, suggesting that establishment of chemoresistance model is being efficient. In addition, microscopic analyses indicated morphological changes on HCT-116 5-FUR cells. The cell area and perimeter analysis demonstrated increased cell size of HCT-116 5-FUR compared with HCT-116P. Moreover, HCT-116P showed a cuboid shape similar to a perfect circle whereas circularity was lower on HCT-116 5-FUR cells, indicating a spindle-shaped morphology acquisition, compatible with EMT cells. In addition, HCT-116 5-FUR exhibited multinucleated cells and cytoplasmic vacuoles as expected in chemoresistance cells. Taken together, these preliminary data suggest that the chronic treatment with 5-FU could induce colon cancer drug resistance and changes in cell morphology that could be associated whit EMT acquisition, indicating a possible association between both phenotypes.
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Paola Lugato Baptista
Boa tarde! Obrigada pelo interesse no nosso trabalho! Nós realizamos alguns testes preliminares com a linhagem HT-29 usando a mesma metodologia (tratamento crônico com diferentes doses de 5-FU). No entanto, não prosseguimos com essa linhagem pois uma população muito pequena sobreviveu ao tratamento, e as poucas células que sobreviveram não voltaram a proliferar. Dessa forma, análises morfológicas não foram realizadas. Na literatura, encontramos alguns estudos que utilizaram a linhagem HT-29, no entanto a maioria utilizou tratamento crônico; possivelmente pela baixa tolerância dessa linhagem ao 5-FU. Acredito que para induzir/observar mudanças morfológicas através do tratamento agudo, seria necessário utilizar uma dose subletal do 5-FU.
Se tiver mais alguma dúvida, estou a disposição.
Mateus Rainho
Muito obrigado pela resposta! Fiz esta pergunta pois em um trabalho de 2019 (https://doi.org/10.1016/j.jsbmb.2019.02.003) trabalhou com as células refratárias à linhagem HT-29 em uma super dosagem de 5-FU (6 μg/ml - 46 μM), mas ele não explorou as características morfológicas. Você acredita que uma única super dosagem pode ser menos prejudicial às células HT-29 do que uma doses repetidas clinicamente relevantes? Agradeço desde já a atenção dispendida.
Paola Lugato Baptista
Bom dia! Obrigada pelo seu interesse! Eu acredito que a utilização de dose aguda (superdosagem ou clinicamente relevante) ou dose crônica vai depender do objetivo do trabalho.
Alguns pontos que devemos considerar:
- no artigo que você enviou, eles usam uma dose de 46uM de 5-FU por 24h; se formos considerar o pico de concentração plasmática do 5-FU (em torno de 200uM), a dose que eles utilizaram não poderia ser considerado uma superdosagem; mesmo assim, é uma dose relativamente alta em comparação com as doses que testamos previamente e vimos uma alta taxa de morte celular (o que inviabilizou a realização de outros testes);
- Se o objetivo do trabalho for analisar mudanças morfológicas, acredito que a exposição crônica com doses mais baixas seria mais viável.
-Se o objetivo for analisar morte celular e seus mecanismos, a exposição aguda seria a melhor metodologia.
Estou a disposição para mais informações.