In Vitro Validation of Network Pharmacology Predictions: Ginsenoside Rg3 Inhibits Hepatocellular Carcinoma Cell Proliferation via SIRT2

Abstract

To elucidate the molecular mechanisms underlying the therapeutic activity of ginsenoside Rg3 (Gs-Rg3) in the context of hepatocellular carcinoma (HCC). Methods Relevant databases were searched to identify protein targets that were both dysregulated and implicated in HCC, as well as targeted by Gs-Rg3. Generation of a protein-protein interaction network facilitated the selection of connected nodes for the construction of a shared disease- and drug-target interaction network model, and topological analysis identified the most highly connected nodes. Targets were annotated with their associated Gene Ontology terms, followed by Kyoto Encyclopedia of Genes and Genomes biological pathway enrichment analysis. In vitro experiments using 2 hours CC cell lines (Bel-7402 and HCCLM3) were performed to investigate the impact of Gs-Rg3 on cell proliferation, viability, cell cycle, cyclin D1 and sirtuin 2 (SIRT2) levels, and global cellular histone acetylation (specifically H3K18ac and H4K16ac). Results Network pharmacology suggested that Gs-Rg3 synergistically targets multiple proteins and pathways relevant to HCC pathogenesis, including those involved in cell cycle and proliferation. In vitro experiments confirmed that Gs-Rg3 dose-dependently inhibits cell proliferation and viability; induces G1 phase cell cycle arrest; decreases cyclin D1, cyclin-dependent kinase 2 (CDK2), and SIRT2 levels; and enhances global H3K18ac and H4K16ac. Conclusions Hypotheses derived from the network analysis were confirmed in vitro. Gs-Rg3 induces G1 phase cell cycle arrest, concomitant with decreased cyclin D1 and CDK2 levels, suggesting a possible mechanism for inhibiting proliferation. In addition, Gs-Rg3 decreases SIRT2 levels, concomitant with enhanced global H3K18ac and H4K16ac. These findings provide a theoretical basis and a support for further preclinical study of the safety and antineoplastic molecular mechanisms of Gs-Rg3, with the goal of eventual clinical translation.

Chinese Herbal Medicine for Primary Liver Cancer Therapy: Perspectives and Challenges

“…Ginsenoside Rg3 significantly inhibits the growth of a variety of liver cancer cell lines, including HepG2, SK-Hep1, MHCC-97L, MHCC-97H, SMMC-7721, and BEL-7404, and effectively inhibits the migration and invasion of liver cancer cells by upregulating ARHGAP9 protein expression ( Sun et al, 2019 ). Furthermore, ginsenoside Rg3 has been reported to induce G1 arrest during the cell cycle progression of HCC cells, which is associated with decreased expression of CDK2, cyclin D1, PCNA, and SIRT2 and increased expression of H3K18ac and H4K16ac ( Li et al, 2018 ; Shan et al, 2019 ; Zheng Q. et al, 2021 ). In addition, ginsenoside Rg3 not only reduces the expression and activity of NHE1 by inhibiting EGF-EGFR-ERK1/2-HIF-1 alpha signaling ( Li et al, 2018 ) but also activates the mitochondria-mediated caspase cascade, leading to the loss of the mitochondrial membrane potential.…”