Network Pharmacology Analysis and In Vitro Validation of the Active Ingredients and Potential Mechanisms of Gynostemma Pentaphyllum Against Esophageal Cancer

Abstract

Background: Esophageal cancer (EC) is one of the deadliest malignancies worldwide. Gynostemma pentaphyllum Thunb. Makino (GpM) has been used in traditional Chinese medicine as a treatment for tumors and hyperlipidemia. Nevertheless, the active components and underlying mechanisms of anti-EC effects of GpM remain elusive.

Objective: This study aims to determine the major active ingredients of GpM in the treatment of EC and to explore their molecular mechanisms by using network pharmacology, molecular docking, and in vitro experiments.

Methods: Firstly, active ingredients and potential targets of GpM, as well as targets of EC, were screened in relevant databases to construct a compound-target network and a protein-protein interaction (PPI) network that narrowed down the pool of ingredients and targets. This was followed by gene ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, molecular docking, ADME and toxicity risk prediction, cell viability assays, in vitro scratch assays, Transwell cell invasion assays, and Western blotting analysis were subsequently applied to validate the results of the network analysis.

Results: The screening produced a total of 21 active ingredients and 167 ingredient-related targets for GpM, along with 2653 targets for EC. The PPI network analysis highlighted three targets of interest, namely AKT1, TP53, and VEGFA, and the compound-target network identified three possible active ingredients: quercetin, rhamnazin, and isofucosterol. GO and EKGG indicated that the mechanism of action might be related to the PI3K/AKT signaling pathway as well as the regulation of cell motility and cell migration. Molecular docking and pharmacokinetic analyses suggest that quercetin and isoprostanoid sterols may have therapeutic value and safety for EC. The in vitro experiments confirmed that GpM can inhibit EC cell proliferation, migration, and invasion and suppress PI3K and AKT phosphorylation.

Conclusion: Our findings indicate that GpM exerts its anti-tumor effect on EC by inhibiting EC cell migration and invasion via downregulation of the PI3K/AKT signaling pathway. Hence, we have reason to believe that GpM could be a promising candidate for the treatment of EC.

Keywords: Gynostemma pentaphyllum; KYSE- 150.; PI3K/AKT; esophageal cancer; molecular docking; network pharmacology.

Fig. (1)

Flow chart of the research conducted in the present study.

Fig. (2)

GpM and EC network analyses. (A) Venn diagram of GpM and EC targets. (B) Compound-target network built from the co-owned targets in (A). Targets are represented by the round nodes in the middle, with bigger ones having more connections. (C) PPI network generated from the co-owned targets in (A). (D) A cluster of key targets retrieved from (C), with node size and color being proportional to the degree of interaction.

Fig. (3)

GO and KEGG enrichment analyses of GpM in the treatment of EC. (A) GO-BP analysis; (B) GO-CC analysis; (C) GO-MF analysis; (D) KEGG enrichment analysis; (E) Distribution of GpM target proteins on the PI3K-AKT pathway. Highlighted nodes are the identified target proteins of GpM for EC treatment, while unhighlighted nodes are irrelevant targets in the pathway.

Fig. (4)

Molecular docking shows the binding of GpM active components to the three targets of interest.

Fig. (5)

GpM inhibits in vitro proliferation, migration, and invasion of KYSE-150 cells in a dose-dependent manner. (A) Effects of GpM on cell viability at different concentrations, assessed by CCK-8 assays. (B) Effects of GpM on cell migration during wound healing at different concentrations, assessed by scratch assays. (C) Effects of GpM on cell invasion at different concentrations, assessed by transwell invasion assays. The data are represented as means ± SD. *P<0.05;**P<0.01;***P<0.001;****P<0.0001.

Fig. (6)

Western blot analysis of the expression of the PI3K/AKT signaling pathway. (A) Representative blots showing the effect of low (0.55 mg/mL) or high (1.1 mg/mL) aqueous extract on the expression level of PI3K/AKT signaling pathway in KYSE-150 cells. (B) Quantified results of protein levels, which were adjusted to corresponding β-Actin protein level and expressed as fold change relative to control (mean + S.D., n = 3). Differences among all groups were determined by Student's t-test, *p < 0.05, **p < 0.01, as compared with vehicle control.