*2.10. Genistein Inhibits Gene Expression of FAK, Paxillin, Snail, and Twist*

After confirming that genistein inhibits the activation of the focal adhesion pathway, we next investigated whether genistein inhibits FAK and paxillin gene expression levels. qRT-PCR results indicated that 50–100 μM genistein strongly downregulated paxillin and FAK mRNA expression. In addition, we analyzed the expression of other genes of interest. Snail is a transcription factor that regulates the expression of E-cadherin. Twist is closely associated with cervical cancer progression [29–31]. Our results showed that high doses of genistein (50–100 μM) inhibited Snail and Twist expression (*p* < 0.01; *p* < 0.05) (Figure 13). These results confirmed that genistein inhibits migration and invasion via Snail- and Twist-mediated EMT (Epithelial Mesenchymal Transition).

**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.

#### **3. Discussion**

The effects of genistein (alone or in combination with anti-cancer drugs) on growth regulation, angiogenesis, and metastasis have been extensively investigated in several different tumors [32–34]. Multiple molecular mechanisms have been implicated in its actions, including inhibition of the NF-κB, Wingless, and integration 1 beta-catenin (Wnt/betacatenin); mitogen-activated protein kinase (MAPK); and phosphoinositide 3 kinase/Akt (PI3K/Akt) signaling pathways [35]. However, evidence regarding the effects of genistein on cervical cancer is limited. Therefore, the underlying mechanism of genistein's action against human cervical cancer, especially its anti-metastatic potential, remains unknown. The present study was designed to elucidate the mechanism of genistein's action against cervical cancer.

We first used a network pharmacological approach to predict potential targets of genistein's action against human cervical cancer in silico. We identified 10 hub targets of genistein treatment: FN1; TIMP1, GAS6, IL6, C3, IGFBP3, IGFBP4, IGFBP1, CST3, and SPP1. FN1 encodes fibronectin, which monitors proliferation and metastasis by regulating the FAK signaling pathway in cervical cancer cells [12]. Previous in silico studies have identified the focal adhesion pathway as a key pathway in cervical cancer [36]. Moreover, the focal adhesion pathway is already considered a potential target in the treatment of highly invasive cancers [37–39]. Focal adhesion kinase (FAK) is an intracellular tyrosine kinase and plays an important role in the regulation of ECM integrin signaling [40–43]. Evidence is accumulating that demonstrates that FAK promotes tumorigenesis through a wide range of cellular processes, including proliferation, survival, metastasis, angiogenesis, epithelial–mesenchymal transition (EMT), cancer stem cell activities, and the metabolism of glucose, lipids, and glutamine [12,44,45]. In our in silico study, focal adhesion was identified using KEGG pathway enrichment analysis as one of the targeted pathways that regulate the anti-cancer effects of genistein (Figures 4 and 11). Considering all the evidence, the FAK pathway was selected as the main pathway for experimental validation.

Phenotypic studies revealed that genistein decreased the proliferation of HeLa cells. This observation is consistent with a previous study indicating that genistein demonstrates cytotoxic properties in numerous different cell types [17,20]. Moreover, genistein is reported to inhibit the proliferation of HeLa, CaSki, and C33 cell lines, and HeLa cells are more sensitive to genistein [27]; therefore, we used the HeLa cell line for our mechanism study. According to our results, genistein also dose-dependently inhibited the HeLa cells' adhesion and metastasis. The inhibition of adhesion is consistent with genistein's inhibition of focal adhesion pathway activation and genistein's inhibition of vimentin and β-catenin expression. Thus, our results provide evidence that genistein suppresses cervical cancer cell metastasis by regulating the FAK/paxillin pathway. Genistein has previously been shown to influence cervical cancer by altering epigenetic modulatory signatures and inducing apoptosis [21,23]. Our results provide strong evidence that genistein inhibits both the activation of the FAK/paxillin pathway and FAK and paxillin gene expression in cervical cancer cells. These results are consistent with the published data and our previously obtained data [17]. We showed that genistein inhibited p-FAK after only 10 min treatment and had a stable inhibitory effect after 24 h treatment in melanoma cells. In human cervical cells, genistein exerts the same behavior. Furthermore, we have previously reported that high concentrations of genistein strongly decrease Snail expression in melanoma cells [17]. Snail is an important transcription factor regulating the process of EMT; the overexpression of Snail in several tumor tissues is associated with metastasis and recurrence [46]. Here, we show that genistein also inhibits Snail expression in human cervical cancer cells.

The role of genistein in cervical cancer was also explored by RNA expression profiling. Thus, the influence of genistein on the regulation of RNA transcription, processing, and splicing in cervical cancer cells was elucidated. The top KEGG pathways enriched using the downregulated DEGs included the spliceosome, ribosome, cell cycle, RNA transport, ribosome biogenesis in eukaryotes, ubiquitin-mediated proteolysis, RNA polymerase, steroid biosynthesis, viral carcinogenesis, central carbon metabolism in cancer, microRNAs

in cancer, focal adhesion, cellular senescence, and thyroid hormone signaling pathways. Using RNA sequencing analysis, we were able to confirm our in silico predictions that focal adhesion was a target pathway. In addition to inhibiting focal adhesion, genistein downregulated the expression of PKC, Src, Shc, Pak, Actinin, Talin, and ILK (Figure 11). Protein kinase C (PKC) is a serine-threonine kinase found in most cell types, where it exerts a strong influence on signal transduction events. Elevated expression of PKC has been highly associated with several human cancers, and inhibition of PKC signaling retards the growth and invasion of cervical cells [47]. The proto-oncogene tyrosine-protein kinase Src (Src) is involved in cell growth, differentiation, migration, and survival. FAK and Src are recruited upon integrin activation to form the FAK–Src complex, and this complex phosphorylates downstream adaptor proteins such as paxillin. The activated FAK–Src complex has an essential role in controlling cell shape and cell motility [48,49].

Our RNA profiling analysis revealed that the downregulated DEGs are involved in the regulation of the cell cycle. This result is consistent with our earlier published data. We previously found that genistein could block the cell cycle arrest of macrophages in the G2/M phase [50]. In addition, genistein strongly downregulated mitochondrial organization and disrupted several metabolic pathways, including the glucose catabolic process, ATP (Adenosinetriphosphate) generation from ADP (Adenosinediphosphate), and multi-organism metabolic processes. According to previous reports, genistein can trigger anti-cancer activity against several cancer cells involved in mitochondrial apoptosis [51–53]. However, in cervical cancer, genistein's action on mitochondrial function has not yet been reported. Therefore, this result may herald a new direction in cervical cancer research.

In conclusion, our study provides evidence that the mode of action of genistein against cervical cancer is comprehensive, from genotype to phenotype. Our results reveal that genistein exerts its anti-proliferation and anti-metastatic activities against cervical cancer by interacting with several key pathways. Genistein inhibits the FAK/paxillin pathway and strongly regulates Twist/Snail-mediated EMT, two pathways related to the progression of cervical cancer. Differing from the dual function of genistein in regulating the metastasis of melanoma cells [17], the predominant inhibitory effects of genistein on cervical cancer cells were identified based on RNA expression profiling and phenotypic studies. The novel mechanisms of action of genistein against cervical cancer identified in this study should prove useful in future research and in clinical applications. Overall, our study provides strong evidence that genistein is a promising chemotherapeutic agent against cervical cancer.
