Integrating experimental and network pharmacology to explore the pharmacological mechanisms of Dioscin against glioblastoma

Abstract

Background: Dioscin (Dio) is an important anti-tumor active component found in the seeds of Livistona chinensis. However, the efficacy and mechanism of Dio in relation to the progression of glioblastoma (GBM) remain unclear.

Materials and methods: Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays, flow cytometry, and Hoechst staining in vitro experiments. A tumor-bearing nude mouse model was employed to further explore the impact of Dio on GBM tumor growth in vivo experiments. Using network pharmacological analysis and molecular docking to predict the potential target proteins and signaling pathways of Dio anti-GBM. The expression of proteins associated with apoptotic pathways was assessed by western blotting.

Results: Dio effectively suppresses the proliferation and promotes apoptosis of U251 cells. In the established tumor-bearing nude mouse model, the anti-cancer activity of Dio was further assessed. Kyoto Encyclopedia of Genes and Genomes enrichment analysis highlighted cancer signaling pathways, including epidermal growth factor receptor (EGFR). Western blot results showed that EGFR phosphorylation and apoptosis gene CASP3 increased with the increase of Dio concentration.

Conclusions: Dio could inhibit the proliferation and promote apoptosis of GBM cells, and play a significant inhibitory role in tumor growth. Dio could affect the phosphorylation of EGFR and then trigger the apoptosis process, resulting in the up-regulation of apoptosis protein CASP3 expression in GBM cells.

Keywords: Dioscin; EGFR; apoptotic pathway; glioblastoma; network pharmacology.

Figure 1

Flow chart of this study.

Figure 2

CCK8, flow cytometry, and Hoechst staining assay. (a) Effects of Dio on the proliferation inhibition rate of U251 cells. (b) Effect of Dio on the apoptosis of U251 cells. (c) After Hoechst dye staining, the apoptosis of U251 cells treated with Dio was observed by fluorescence microscope and identified by red arrows. The magnification was 10×.

Figure 3

Dio inhibited tumor growth in vivo. (a) Tumor growth was monitored by bioluminescence imaging technique, and the tumor growth size and subcutaneous xenograft tumor tissue were photographed. Shown are representative images from at least three independent experiments. (b) Tumor weight was monitored as described in (a). **P ＜ 0.01.

Figure 4

Screening for Dio-related targets and PPI network analysis. (a) Chemical structure of Dio. (b) Venn diagram illustrating the overlap of Dio targets and GBM disease targets. (c) Yellow nodes represent targets with higher degree values. (d) Visualization of interactions among overlapping genes.

Figure 5

GO and KEGG analysis: (a) enrichment analysis of potential targets of Dio treatment of GBM and (b) KEGG pathway.

Figure 6

Molecular docking map of active ingredients with key targets: (a) Dio-EGFR, (b) Dio-CASP3, (c) Dio-AKT, (d) Dio-HSP90AA1, (e) Dio-SRC, (f) Dio-ESR1, (g) Dio-STAT3, (h) Dio-MAPK1, (i) Dio-MAPK8, and (j) Dio-MDM2.

Figure 7

Expression and overall survival of hub genes in GEPIA2 database. (a) Expression of hub genes in GEPIA2 database, the red box plots represent tumor and the gray box plots represent normal. (b) Overall survival of hub genes in GEPIA2 database, the blue represents low expressions and the red represents high expression.

Figure 8

Western blotting analysis of Caspase3, p-EGFR protein, and EGFR after Dio (0, 2, 4, 6 μM) treatment in U251 cells for 24 h.