Identification of potential anti-tumor targets and mechanisms of HuaChanSu injection using network pharmacology and cytological experiments in Breast cancer

HuaChanSu (HCS), i.e., cinobufacini injection, is an anti-tumor agent with anti-tumor effects. However, the mechanism underlying the anti-tumor effect of HCS in breast cancer (BRCA) is still unclear. Therefore, in this study, we determined the effects of HCS on BRCA cells using cytological, comprehensive network pharmacology, and molecular biology approach. The effect of HCS on BRCA cell activity was determined using cytotoxicity assays. A linear fit was performed to calculate the half-inhibitory concentration (IC50) value of HCS. Subsequently, the cells were divided based on the IC50 values for TUNEL and scratch assay into the negative control (NC), high-dose HCS (HCS-H), and low-dose HCS (HCS-L) groups. Subsequently, functional enrichment analysis was performed on potential targets to identify the functions and potential pathways enriched by HCS for BRCA treatment. Molecular docking was performed on HCS active compounds and key proteins. Finally, we performed western blotting (WB) on HCS-treated BRCA cells to determine the expression of key signaling pathways-related proteins. In vitro experiments revealed that HCS attenuated proliferation as well as migration and induced apoptosis of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Subsequently, we identified the potential HCS targets and target genes for BRCA. The 289 core HCS targets against BRCA were screened from the PPI network and functional enrichment analysis. These core targets were primarily enriched in signaling pathways like PI3K-AKT, MAPK chemokines, and others. Important core targets (PIK3CA, PIK3CD, and MTOR) were obtained from the target proteins of the PI3K-AKT pathway involved in BRCA by HCS and confirmed using molecular docking. In addition, a reduction in phosphorylated PI3K, AKT, and the AKT and MTOR expression level was observed in HCS-treated BRCA cells. These results indicate that HCS could be used as a therapeutic approach for treating patients with BRCA.