基于网络药理学与体外实验探讨香荆芥酚诱导鼻咽癌细胞凋亡和周期阻滞的作用机制

Exploration on the mechanisms of carvacrol-induced apoptosis and cell cycle arrest in nasopharyngeal carcinoma cells based on network pharmacology and in vitro experiments

摘要:

目的 探讨香荆芥酚(CV)对鼻咽癌CNE2细胞增殖、凋亡及周期的影响，并探索CV诱导CNE2细胞凋亡和周期阻滞的潜在机制。方法 运用网络药理学预测CV作用于鼻咽癌的潜在靶点，利用R软件对潜在靶点进行基因组百科全书(KEGG)通路富集分析，使用CB-Dock2平台将CV与靶点蛋白进行分子对接，使用Gromacs 2022.3软件将CV与靶点蛋白进行分子动力学模拟。通过集落形成实验和流式细胞术检测CV对CNE2细胞增殖、凋亡及周期的影响，通过Western blot法检测CV对CNE2细胞内相关蛋白表达的影响。结果 经网络药理学预测，CV作用于鼻咽癌存在137个共有靶点，KEGG富集分析结果显示CV作用于鼻咽癌可能涉及磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)等信号通路；分子对接结果显示PI3K、AKT、磷脂酰肌醇3-激酶催化亚基α(PIK3CA)、B细胞淋巴瘤-2(Bcl-2)、半胱天冬酶-3(Caspase-3)、周期蛋白依赖性激酶2(CDK2)与CV具有较好的结合亲和力。分子动力学模拟进一步表明CV与CDK2结合较稳定。与对照组比较，经CV作用后的CNE2细胞增殖能力降低，细胞凋亡能力增加，细胞周期G0/G1期延长，PIK3CA、p-PI3K、p-AKT、Bcl-2和CDK2蛋白表达均明显降低，Bcl-2相关X蛋白(Bax)和Caspase-3蛋白表达均明显升高，差异均具有统计学意义(P均＜0.05)。结论 CV抑制CNE2细胞增殖，并诱导细胞凋亡和阻滞细胞周期于G0/G1期。CV诱导CNE2细胞凋亡和周期阻滞可能与PI3K/AKT信号通路相关的PIK3CA、p-PI3K、p-AKT、Bcl-2、CDK2蛋白表达降低及Bax、Caspase-3蛋白表达升高有关。

Abstract:

Objective To investigate the effects of carvacrol (CV) on the proliferation, apoptosis, and cell cycle of nasopharyngeal carcinoma CNE2 cells, and to explore the potential mechanisms underlying CV-induced apoptosis and cell cycle arrest in CNE2 cells. Methods Network pharmacology was used to predict the potential targets of CV in nasopharyngeal carcinoma. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was performed on these potential targets using R software. Molecular docking between CV and target proteins was conducted using the CB-Dock2 platform, and molecular dynamics simulations of CV and target proteins were performed using Gromacs 2022.3 software. Colony formation assays and flow cytometry were used to assess the effects of CV on the proliferation, apoptosis, and cell cycle. Western blotting was employed to examine the impact of CV on protein expression in CNE2 cells. Results Network pharmacology predictions indicated that CV affected nasopharyngeal carcinoma through 137 common targets. KEGG pathway enrichment analysis suggested that CV might involve several signaling pathways, including phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). Molecular docking results revealed that PI3K, AKT, PIK3CA, Bcl-2, Caspase-3, and CDK2 exhibited strong binding affinities with CV. Molecular dynamics simulations further demonstrated that CV formed a stable binding with CDK2. Compared with the control group, the proliferative capacity of CNE2 cells after CV treatment decreased, the apoptosis ability increased, the G0/G1 phase of the cell cycle was prolonged, and the expressions of PIK3CA, p-PI3K, p-AKT, Bcl-2, and CDK2 proteins all significantly decreased, while the protein expressions of Bax and Caspase-3 significantly increased. All the differences were statistically significant (all P<0.05). Conclusion CV inhibits the proliferation of CNE2 cells, induces apoptosis, and blocks the cell cycle at the G0/G1 phase. The apoptosis and cell cycle arrest induced by CV in CNE2 cells may be associated with the decreased expressions of PIK3CA, p-PI3K, p-AKT, Bcl-2, and CDK2 proteins, as well as the increased expressions of Bax and Caspase-3 proteins, which are related to the PI3K/AKT signaling pathway.