Fig 1.
HCS reduced the viability of MCF-7 and MDA-MB-231 cells.
(A, B) Viability rates of MCF-7 cells treated with the indicated concentrations of HCS for 48 h (C, D) Viability rates of MDA-MB-231 cells treated with the indicated concentrations of HCS for 48 h. The IC50 values of HCS were calculated by plotting dose-viability curves. (E-H) Representative images (200x) and quantification of TUNEL-positive MCF-7 (E, G) and MDA-MB-231 (F, H) in the indicated groups. **P < 0.01, ***P < 0.001, ****P < 0.0001. The data are represented as mean ± SD (n = 3). HCS, HuaChanSu injection; CCK-8, Cell Counting Kit 8; IC50, half-inhibitory concentration.
Fig 2.
HCS inhibited MCF-7 and MDA-MB-231 cell migration.
The extent of wound coverage in MCF-7 (A) and MDA-MB-231 (B) cells in the NC, HCS-L, and HCS-H groups at 0 h and 24 h. **P < 0.01, ***P < 0.001, ****P < 0.0001. The data are presented as mean ± SD (n = 3). HCS, HuaChanSu injection; NC, negative control; HCS-H, high-dose HCS group; HCS-L, low-dose HCS group.
Fig 3.
Construction of BRCA-HCS-active component-target interaction network and PPI network.
(A) Venn diagram showing the intersecting disease targets and HCS targets. (B) The BRCA-HCS-active compound-target interaction network. (C) PPI network of potential target genes. (D) © top 30 potential core target genes based on the Degree algorithm in the cytoHubba plug-in. BRCA, breast cancer; HCS, HuaChanSu injection; PPI, protein-protein interaction.
Fig 4.
Functional enrichment analysis and molecular docking.
(A) Bubble diagram showing the significantly enriched GO terms. (B) Bubble diagram showing the significantly enriched KEGG pathways. (C) HCS target proteins involved in the PI3K-Akt signaling pathway in BRCA (marked in red). (D) Heatmap showing the Vina binding energy values of 16 active compounds and 2 positive chemotherapeutic drugs (marked in blue) with three key target proteins. Black panels indicate absence of any regulatory relationship between the compounds and the target site. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BRCA, breast cancer.
Fig 5.
The three compounds with the lowest affinity score were docked with MTOR.
Binding poses and binding sites of MTOR complexed with ψ-bufarenogin (A), cinobufagin (B), and cinobufotalin (C).
Fig 6.
HCS inactivates the PI3K-Akt/mTOR signaling pathway in BRCA cells.
(A) Immunoblot showing the expression levels of PI3K pathway proteins in the control and HCS-treated MDA-MB-231 cells. GAPDH was used as the control. (B) Comparison of the expression levels of proteins in the NC, HCS-L, and HCS-H groups. All experiments were performed in triplicates. *P < 0.05, ****P < 0.0001. The data are presented as mean ± SD (n = 3). HCS, HuaChanSu injection; NC, negative control; HCS-H, high-dose HCS group; HCS-L, low-dose HCS group.