Genistein Inhibits Proliferation and Metastasis in Human Cervical Cancer Cells through the Focal Adhesion Kinase Signaling Pathway: A Network Pharmacology-Based In Vitro Study in HeLa Cells

Abstract

Previous studies have provided evidence that genistein exerts a therapeutic effect on different tumor cells. However, the mechanism of action of genistein against cervical cancer cells remains largely unknown. The aim of this study was to comprehensively decipher the anti-metastatic effect and molecular mechanism of genistein action on cervical cancer cells. We developed an integrated strategy from genotype to phenotype, combining network pharmacology and a transcriptome screening approach, to elucidate the underlying mechanism of action of genistein against human cervical cancer cells. In silico studies predicted that the focal adhesion pathway may be an important signaling cascade targeted by genistein treatment. Using RNA sequencing analysis, representative genes of the focal adhesion pathway were demonstrated to be significantly downregulated. Phenotypic studies revealed that genistein demonstrated strong anti-proliferative and anti-metastatic activity in HeLa cells. Moreover, genistein modulated this activity in a concentration-dependent manner. Genistein also inhibited both the activation and gene expression of FAK (Focal Adhesion Kinase) and paxillin. In addition, vimentin and β-catenin protein expression, and Snail and Twist gene expression, were strongly inhibited by genistein. Our findings provide strong evidence for a pleiotropic effect of genistein on cervical cancer cells, mediated through the focal adhesion pathway.

Keywords: RNA expression profiling; cervical cancer; genistein; metastasis; network pharmacology.

Figure 1

Venn plot of genes potentially involved in genistein’s action against human cervical cancer. The purple color represents the targets of genistein, and the yellow color represents the genes related to the progression of human cervical cancer.

Figure 2

PPI network of the core genes in genistein’s action against human cervical cancer (dark purple color indicates high degree; grey color indicates low degree).

Figure 3

Hub gene analysis of the PPI network. The color change from yellow to red indicates the increasing importance of related genes in the network. The red color indicates the highest significance.

Figure 4

GO ontology and KEGG pathway enrichment of the core targets. (A) Biological processes and (B) KEGG pathways involved in genistein’s action against human cervical cancer.

Figure 5

Predicted targets of genistein’s action in the focal adhesion pathway. Proteins highlighted in red indicate potential targets of genistein’s action against human cervical cancer.

Figure 6

(A). Effects of genistein treatment on the proliferation of HeLa cells for 24–48 h were detected by CCK−8 assay. (B). Effects of genistein treatment on cell growth were counted by hemocytometer after 24–48 h. (C). Representative images of HeLa cell colony formation after genistein treatment (0–100 μM). The data shown are the average of three replicates; the experiments were performed three times independently. *** p < 0.001, ** p < 0.01, * p < 0.05 compared with solvent control. Scale bar: 200 μm.

Figure 7

Effects of genistein on HeLa cell adhesion. Cells were grown in the presence of different doses of genistein for 24 h, and reseeded for 3 h. Adherent cells were then fixed by PFA (Paraformaldehyde) and stained with crystal violet solution. Absorbance readings were detected at OD570 nm by microplate reader. (A). Percentage of adhesion was then calculated based on the OD value of the adhered cells in the genistein-treated group (compared to control values (100%)). The experiment was performed three times independently, and data shown are the average of all three replicates. (B). Representative images from the three independent experiments. ** p < 0.01 vs. DMSO control; * p < 0.05 vs. DMSO control. Scale bar: 200 μm.

Figure 8

Effect of genistein on the mobility of HeLa cells. (A) Representative images of wound-healing assay. (B) Cell migration was calculated by measuring the distances from the wound edges in each treatment group using Image J software. Cell migration activity (as a percentage) was calculated from the migration distances in the genistein treatment group (compared to the control (100%)). The experiment was repeated three times. ** p < 0.01 vs. DMSO control. * p < 0.05 vs. DMSO control; Scale bar: 200 μm.

Figure 9

Effect of genistein on the migration and invasion activities of HeLa cells. Cells were seeded on membranes and co-cultured with different doses of genistein for 24 h. (A) Cells that migrated to the lower surface of the filter were fixed and stained with crystal violet, and then photographed by an inverted microscope at ×100. (B) Cell invasion was assessed by following cell movement through the Matrigel to the lower surface of the filter. Invading cells were fixed, stained with crystal violet, and photographed under an inverted microscope at ×100. (C) Cell migration was quantified from at least three images randomly using Image J software. (D) Cell invasion was quantified from at least three images randomly using the Image J software. The experiment was repeated three times. ** p < 0.01 vs. DMSO control. * p < 0.05 vs. DMSO control; scale bar: 200 μm.

Figure 10

(A) Volcano plot of DEGs identified following genistein treatment (compared with control). Grey dots, genes with no significant difference in expression; blue dots, downregulated genes; and orange dots, upregulated genes. Fold-change was calculated using gene-normalized expression of the genistein group/gene-normalized expression of the control group. Differences in expression with a p value < 0.05 and a Log₂ (fold change) > 1 were considered statistically significant. (B,C) Gene ontology enrichment of up- and downregulated DEGs after genistein treatment. (D) KEGG enrichment of upregulated DEGs after genistein treatment.

Figure 11

(A−D) Representative enrichment of gene signatures in genistein and control group by gene set enrichment analysis (GESA). Representative enriched gene sets are shown (FDR q value < 0.05). (E−H). Heatmap of the representative DEGs between genistein and control group in parallel with GESA analysis.

Figure 12

Effect of genistein on the focal adhesion protein expression in HeLa cells. (A) Cells were grown with or without different doses of genistein for 30 min. The expression levels of specific proteins were detected by Western blot analysis. GAPDH was used as a control. All first antibodies were used at a dilution of 1:1000. Secondary antibodies were used at a concentration of 1:3000. (B–E) Integrated optical intensity of the bands was determined by Image J software (https://imagej.net, accessed on 3 January 2023). The experiment was repeated three times. ** p < 0.01 vs. DMSO control.

Figure 13

Effect of genistein on specific gene expression in HeLa cells. (A) Genistein inhibited FAK and paxillin, and (B) Snail and Twist mRNA expression in HeLa cells. Gene expression was analyzed by qRT-PCR. mRNA relative expression levels were evaluated using the 2^−△△Ct method. GAPDH was used as an internal control. The experiment was repeated three times. ** p < 0.01 vs. DMSO control; * p < 0.05 vs. DMSO control.