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Polymethoxylated Flavones Target Cancer Stemness and Improve the Antiproliferative Effect of 5-Fluorouracil in a 3D Cell Model of Colorectal Cancer

1
iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
2
Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, E.P.E (IPOLFG, EPE), 1099-023 Lisboa, Portugal
3
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal
4
Faculdade de Farmácia da Universidade de Lisboa, Av das Forças Armadas, 1649-019 Lisboa, Portugal
5
Laboratory of Foodomics, Institute of Food Science Research (CIAL, CSIC), Calle Nicolás Cabrera 9, 28049 Madrid, Spain
6
Molecular Nutrition and Metabolism, Institute of Food Science Research (CIAL, CSIC) Calle Nicolás Cabrera 9, 28049 Madrid, Spain
*
Authors to whom correspondence should be addressed.
Nutrients 2019, 11(2), 326; https://doi.org/10.3390/nu11020326
Received: 4 January 2019 / Revised: 26 January 2019 / Accepted: 30 January 2019 / Published: 2 February 2019
(This article belongs to the Special Issue Nutrition and Colorectal Cancer)
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PDF [2713 KB, uploaded 20 February 2019]
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Abstract

Polymethoxylated flavones (PMFs) from citrus fruits are reported to present anticancer potential. However, there is a lack of information regarding their effect on cancer stem cell (CSC) populations, which has been recognized as responsible for tumor initiation, relapse, and chemoresistance. In this study, we evaluated the effect of an orange peel extract (OPE) and its main PMFs, namely, nobiletin, sinensetin, tangeretin, and scutellarein tetramethylether in targeting cell proliferation and stemness using a 3D cell model of colorectal cancer composed of HT29 cell spheroids cultured for 7 days in stirred conditions. Soft agar assay, ALDH1 activity, and relative quantitative gene expression analysis of specific biomarkers were carried out to characterize the stemness, self-renewal, and mesenchymal features of HT29 cell spheroids. Then, the impact of OPE and PMFs in reducing cell proliferation and modulating cancer stemness and self-renewal was assessed. Results showed that, when compared with monolayer cultures, HT29 cell spheroids presented higher ALDH1 activity (81.97% ± 5.27% compared to 63.55% ± 17.49% for 2D), upregulation of CD44, PROM1, SOX9, and SNAI1 genes (1.83 ± 0.34, 2.54 ± 0.51, 2.03 ± 0.15, and 6.12 ± 1.59 times) and high self-renewal capability (352 ± 55 colonies compared to 253 ± 42 for 2D). Incubation with OPE (1 mg/mL) significantly inhibited cell proliferation and modulated cancer stemness and self-renewal ability: colony formation, ALDH1 activity, and the expression of cancer stemness biomarkers PROM1 and LGR5 were significantly reduced (0.66 ± 0.15 and 0.51 ± 0.14 times, respectively). Among all PMFs, tangeretin was the most efficient in targeting the CSC population by decreasing colony formation and the expression of PROM1 and LGR5. Scutellarein tetramethylether was shown to modulate markers of mesenchymal/metastatic transition (increasing CDH1 and reducing ZEB1 and SNAI1) and nobiletin was capable of downregulating PROM1 and SNAI1 expression. Importantly, all PMFs and OPE were shown to synergistically interact with 5-fluorouracil, improving the antiproliferative response of this drug. View Full-Text
Keywords: orange peel extract; polymethoxylated flavones; tangeretin; scutellarein tetramethylether; synergistic interactions; 5-fluorouracil; colorectal cancer; cancer stem cells; 3D cell model orange peel extract; polymethoxylated flavones; tangeretin; scutellarein tetramethylether; synergistic interactions; 5-fluorouracil; colorectal cancer; cancer stem cells; 3D cell model
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Pereira, C.V.; Duarte, M.; Silva, P.; Bento da Silva, A.; Duarte, C.M.M.; Cifuentes, A.; García-Cañas, V.; Bronze, M.R.; Albuquerque, C.; Serra, A.T. Polymethoxylated Flavones Target Cancer Stemness and Improve the Antiproliferative Effect of 5-Fluorouracil in a 3D Cell Model of Colorectal Cancer. Nutrients 2019, 11, 326.

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