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

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.

Phytochemical Composition, Antioxidant and Antiproliferative Activities of Defatted Sea Buckthorn (Hippophaë rhamnoides L.) Berry Pomace Fractions Consecutively Recovered by Pressurized Ethanol and Water

“…Antiproliferative effect of SBP extracts and standard compounds was evaluated in HT29 cells as described elsewhere [30]. The cells were seeded at a density of 1 × 10 4 cells/well in 96-well plates.…”

Precision Nutrition and Cancer Relapse Prevention: A Systematic Literature Review

“…Other extracts with identified effects on cell growth, tumor progression and metastasis in colorectal cancer are watercress extract, rich in phenethyl isothiocyanate [86]; ginseng extract, rich in ginsenoside Rg3 [79]; and isodon extract, rich in flexicaulin A [81]. Importantly, orange peel extract, rich in nobiletin, sinensetin, scutellarein tetramethylether, and tangeretin, exerts a synergistic interaction with 5-fluorouracil in colorectal cancer, modulating EMT transition, inhibiting cell proliferation and modulating cancer stemness, demonstrating a significant potential use of this combination in relapse preventive therapies [82]. In any case, further research needs to be done in order to identify molecular mechanisms regarding the effects of these extracts.…”

Valorization of European Cranberry Bush (Viburnum opulus L.) Berry Pomace Extracts Isolated with Pressurized Ethanol and Water by Assessing Their Phytochemical Composition, Antioxidant, and Antiproliferative Activities

“…Cytotoxicity was assessed using confluent and non-differentiated Caco-2 cells as a model of the human intestinal epithelium, as previously described [ 29 ]. Briefly, Caco-2 cells were incubated with the samples diluted in the culture medium (RPMI supplemented with 0.5% FBS).…”