Azbazdar Y et al. (2024), Interactions between genistein and Wnt pathway ...

XB-ART-60768

Heliyon 2024 Jun 15;1011:e32243. doi: 10.1016/j.heliyon.2024.e32243.

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Interactions between genistein and Wnt pathway in colon adenocarcinoma and early embryos.

Azbazdar Y , Sosa EA , Monka J , Kurmangaliyev YZ , Tejeda-Muñoz N .

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The Wnt signaling pathway is one of the most ancient and pivotal signaling cascades, governing diverse processes in development and cancer regulation. Within the realm of cancer treatment, genistein emerges as a promising candidate due to its multifaceted modulation of various signaling pathways, including the Wnt pathway. Despite promising preclinical studies, the precise mechanisms underlying genistein's therapeutic effects via Wnt modulation remain elusive. In this study, we unveil novel insights into the therapeutic mechanisms of genistein by elucidating its inhibitory effects on Wnt signaling through macropinocytosis. Additionally, we demonstrate its capability to curtail cell growth, proliferation, and lysosomal activity in the SW480 colon adenocarcinoma cell model. Furthermore, our investigation extends to the embryonic context, where genistein influences gene regulatory networks governed by endogenous Wnt pathways. Our findings shed light on the intricate interplay between genistein, Wnt signaling, membrane trafficking, and gene regulation, paving the way for further exploration of genistein's therapeutic potential in cancer treatment strategies.

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Species referenced: Xenopus Xenopus laevis
Genes referenced: otx2 sox2
GO keywords: lysosome [+]
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	Fig. 1. Genistein Inhibits Activation of Wnt Pathway and Cell Proliferation (A-F) Genistein blocks activation of the Wnt pathway by LiCl and inhibits β-catenin stabilization in HEK 293 cells (B) Control (untreated cells). (C) Genistein treatment (500 μM for 3 h). (D) LiCl treatment (40 mM for 3 h) results in increased β-catenin levels. (E) Genistein + LiCl treatment. No increase in β-catenin levels. (F) BAR Luciferase assay of β-catenin activity levels for the same experiments as inB-E. (G) Cell proliferation assay in SW480 cells was reduced after genistein treatment (250 μM). All experiments with cultured cells were repeated in at least 3 biological replicates. Red: β-catenin; blue: nuclei. Scale bar, 10 μm. Error bars denote SEM (n ≥ 3) (P < 0.05,P < 0.01, ***P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 2. Genistein Inhibits Macropinocytosis in Colon Cancer Cells (A-D) Illustration of the cell membrane with genistein inhibiting macropinocytosis. (B–B′) TMR-dx (red, 1 mg/mL) uptake in SW480 cells after 1 h of incubation (control) (C–C′) Same as B–B′ after overnight genistein treatment (250μM). The TMR-dx uptake was reduced. (D) TMR-dx uptake quantification for B–C’. Percentage of cells with detectable TMR-dx signal. (E–K) Genistein reduces cell growth in SW480 spheroids (F) SW480 spheroids at 96 h of inverted drop culture visualized in bright-field (see Methods). (G) SW480 spheroids with the same conditions as F after the overnight genistein treatment (250 μM), the sizes of spheroids in 3D culture were reduced. (H) Quantification of the spheroid area from F and G. (I) TMR-dx (red, 1 mg/mL) uptake in spheroids. (J) After the overnight genistein treatment (250 μM), the TMR-dx uptake was reduced in cells treated with the same conditions as I. (K) TMR-dx uptake quantification for I and J. All experiments with cultured cells were repeated in at least 3 biological replicates.Eight spheroids were plated per replicate. Scale bars, B–C′10 μm; F-J 500 μm. Error bars denote SEM (n ≥ 3) (P < 0.01, *P < 0.001).). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. Genistein Reduces Lysosome Formation and β-catenin Levels in Colon Cancer Cells (A) Genistein reduces multivesicular body formation (MVB) in SW480 cells. (B–B″) Untreated (control) SW480 cells have high levels of lysosomes visualized by MVB marker CD63 (green) and high levels of nuclear β-catenin (red). (C–C″) Cells with identical treatment as (B)-(B″) after the overnight genistein treatment (250 μM). Genistein treatment reduces both CD63 and β-catenin and CD63 levels. All experiments with cultured cells were repeated in at least 3 biological replicates. Blue: nuclei. Scale bar, 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 4. Genistein Inhibits Head Formation in Early Embryos via Wnt signaling (A) Xenopus embryos at tadpole stage. A, anterior; P, posterior. (B) Same as A after genistein treatment (500 μM) from the 4-cell stage until the 9.5 stage (early gastrula). Genistein treatment results in ventralized embryos. (C and D) Same conditions as A and B. A pan-neuronal marker Sox2 is visualized by in situ hybridization. (C′ and D′) Same as C and D. Frontal view showing the reduction in the head formation (white arrow). (E–F) Same conditions as A and B. A forebrain/midbrain marker Otx2 is visualized by in situ hybridization. All experiments were repeated five times. Total numbers of embryos analyzed for each condition, and percentage of embryos with reported phenotypes: A = 130, 100 %; B = 138, 93 %; C = 50, 100 %; D = 62, 91 %; E = 60, 100 %; F = 65; 92. Scale bars, 500 μm.
	Fig. 5. Genistein Treatment Has Broad Effects on Gene Regulation in Early Embryos (A) Experimental design. Xenopus embryos were treated with different drugs at the 4-cell stage. Total RNA was extracted at the 9.5 stage and used for RNA sequencing. Three replicates with three embryos per replicate were used for each condition. (B) A heatmap of expression patterns of top DEGs in each condition. For genistein, we show the top 30 upregulated and downregulated DEGs. For BIO and Chiron, we show the top 15 upregulated DEGs (few genes were downregulated; see D and F). The pink and blue bars represent up-regulated and down-regulated genes, respectively. (C) Volcano plot of the differential expression analysis between control and genistein treatments. Log2-fold-change values (x-axis) and adjusted Pvalues (y-axis) are shown for each gene. Differentially expressed genes (DEGs, adjusted P < 0.05 and log2-fold-change >1) are in red. Gene names are shown for top DEGs. (D) Gene Ontology enrichment analysis of DEGs from panel B. The x-axis summarizes the fold enrichments of DEGs for the biological process on the y-axis. Significance levels were computed to adjusted for multiple hypotheses testing via the -log10 of the false-discovery rate (FDR ≤0.05). (E and F) Same as B and C for the comparison of control and BIO (30 mM, a specific inhibitor of GSK-3) (G and H) Same as B and C for comparing control and Chiron (CHIR 99021, 60 μM). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

References [+] :

Albrecht, GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery. 2020, Pubmed, Xenbase