β-榄香烯通过调节 PI3K/Akt/NF-κB 通路影响 MMP9 的表达抑制食管鳞癌细胞迁移。
β-elemene Suppresses Migration of Esophageal Squamous Cell Carcinoma by Modulating Expression of MMP9 through the PI3K/Akt/NF-κB Pathway.
机构信息
Department of Gastroenterology, Cangzhou Central Hospital, Cangzhou, Hebei, 061001, China.
Department of Gastroenterology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050011, China.
出版信息
Comb Chem High Throughput Screen. 2023;26(13):2304-2320. doi: 10.2174/1386207326666230303120514.
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.
背景与目的
β-榄香烯是一种具有广谱抗癌活性的植物衍生药物。研究发现，β-榄香烯能够抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡，并抵抗肿瘤细胞迁移和侵袭。食管癌是一种常见的消化道恶性肿瘤，在食管癌的治疗中取得了一定进展，包括β-榄香烯的应用，但抗迁移的机制尚不清楚。PI3K/Akt/NF-κB/MMP9 信号通路参与肿瘤细胞增殖、迁移、细胞外基质（ECM）和基底膜（BM）降解的调节。本研究旨在通过生物信息学、网络药理学和分子对接方法，探讨β-榄香烯对食管鳞状细胞癌（ESCC）迁移的影响及其相关机制。
方法
本研究通过 GeneCards 和 BATMAN-TCM 数据库结合基因表达综合数据库（GEO）数据库（GSE17351）筛选 ESCC 的差异表达基因（DEGs）。对这些 DEGs 进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路分析，以鉴定基因的功能和相关通路。使用 STRING 数据库构建这些 DEGs 的蛋白质-蛋白质相互作用（PPI）网络。根据度值原则，利用 Cytoscape 中的 CytoHubba 插件筛选出 5 个枢纽基因，并通过癌症基因组图谱（TCGA）中的 UALCAN 数据库验证其表达。通过分子对接确定与 MMP9 结合能最强的枢纽基因。采用划痕愈合实验评估迁移能力，RT-PCR 检测 ESCC 组织中迁移相关 mRNA 的含量，Western blot 检测 ESCC 组织中 Akt、NF-κB 和 MMP9 的表达率。
结果
共获得 71 个靶基因，主要涉及表皮发育和细胞外基质分解等生物学过程。此外，还验证了包括 PI3K/AKT 信号通路和黏附斑在内的关键通路受到β-榄香烯的调控。β-榄香烯与 MMP9 之间表现出显著的结合亲和力，对接评分高达-6.56 kcal/mol。ESCC 组织中 Akt、NF-κB 和 MMP9 的表达水平明显高于正常组织。Western blot 检测显示，β-elemene 特异性降低 Akt 的磷酸化水平及其下游靶分子 NF-κB，从而降低 ESCC 中包括 MMP9 在内的靶蛋白水平。划痕愈合实验表明β-elemene 抑制 ESCC 细胞的迁移。RT-PCR 结果发现，β-elemene 组 Akt、NF-κB 和 MMP9 的 mRNA 表达水平明显低于对照组。然而，SC79 的应用部分逆转了β-elemene 的作用。
结论
综上所述，本研究提示β-elemene 对 ESCC 的抗肿瘤迁移作用与抑制 PI3K/Akt/NF-κB/MMP9 信号通路有关，为进一步合理临床应用提供了理论参考。
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