β-elemene Suppresses Migration of Esophageal Squamous Cell Carcinoma by Modulating Expression of MMP9 through the PI3K/Akt/NF-κB Pathway

Abstract

Background and objective: β-elemene is a plant-derived drug with broad-spectrum anticancer activity. Studies have found that β-elemene can inhibit tumor cell proliferation, induce tumor cell apoptosis, and resist tumor cell migration and invasion. Esophageal cancer is a common digestive tract malignant tumor. Progress has been made in the treatment of esophageal cancer, including the use of β-elemene, but the mechanism of anti-migration is unclear. PI3K/Akt/NF- κB/MMP9 signaling pathway is involved in the regulation of tumor cell proliferation, migration, extracellular matrix(ECM), and basement membrane(BM) degradation. This study aims to investigate the effect of β-elemene on the migration of esophageal squamous cell carcinoma (ESCC) and its related mechanisms by bioinformatics, network pharmacology, and molecular docking methods.

Methods: In this study, the differentially expressed genes (DEGs) of ESCC were screened through GeneCards and BATMAN-TCM databases combined with the Gene Expression Omnibus (GEO) database (GSE17351). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify the functions and related pathways of the genes. The protein-protein interaction (PPI) network of these DEGs was constructed with the STRING database. Five hub genes were screened by CytoHubba plug-in Cytoscape according to the principle of degree value and the expressions of which were validated by the UALCAN database from the Cancer Genome Atlas (TCGA). The hub gene with the strongest binding energy was identified by molecular docking. A wound healing assay was subjected to assess the migration ability. RT-PCR was used to detect the content of migration-related mRNA. Western blotting was performed to examine the expression rates of Akt, NF-κB, and MMP9 in ESCC tissues by β-elemene and SC79.

Results: 71 target genes were obtained which were mainly involved in biological processes such as epidermal development and extracellular matrix decomposition. In addition, critical pathways, including PI3K/AKT signaling pathway and focal adhesion, were verified to be subject to β-elemene regulation. It exhibited marked binding affinity between β-elemene and MMP9 with an excellent docking score of -6.56 kcal/mol. The expression levels of Akt, NF-κB, and MMP9 in ESCC tissues were significantly upregulated compared to normal tissues. Western blot detection demonstrated that β-elemene specifically reduced the phosphorylation level of Akt, and its downstream target molecule NF-κB, thus resulting in reduced levels of their target proteins, including MMP9 in ESCC. A wound healing assay showed β-elemene inhibited the migration of ESCC cells. RT-PCR results found that the mRNA expression of Akt, NF-κB, and MMP9 in the β-elemene group was significantly lower than that in the control group. However, the application of SC79 partially reversed the effect of β-elemene.

Conclusion: In summary, our study suggests that the anti-tumor migration effect of β-elemene on ESCC is associated with the inhibition of PI3K/Akt/NF-κB/MMP9 signalling pathway, and it provides a theoretical reference for further rational clinical application.

Keywords: MMP9; Network pharmacology; PI3K/Akt; esophageal squamous cell carcinoma; migration; β-elemene.