The mechanism of action of limonoids of Citri Reticulatae Semen against MCF-7 breast cancer cells

Limonoids (LMD), particularly limonin, possess significant anti-breast cancer (BC) potential. However, their precise anti- breast cancer mechanisms remain unelucidated. In this study, we aimed to identify potential biomarkers associated with BC development and metastasis and to investigate LMD anti-BC activity. Key LMD components were analyzed using UPLC-QQQ-MS/MS, with various bioinformatics tools utilized for additional analysis. These included ADME and toxicity prediction, Weighted Gene Co-expression Network Analysis (WGCNA), and construction of protein-protein interaction (PPI) and "LMD-Hub-gene-BC" models. A total of 1898 DEGs were obtained including 622 up-regulated and 1276 down-regulated genes. The WGCNA identified 4 significant modules. The 11 hub genes were identified by NTA. The results of enrichment analysis showed that 631 significant GO conditions and 34 significant KEGG pathways were enriched, of which cell cycle, p53 signaling pathway and Prostate cancer might be the main regulatory pathways for its anti-BC. The gene expression and immunohistochemistry of the result suggests that most of the hub genes expression has significant differences (P < 0.05). KM-plotter results revealed that most of the hub genes were associated with prognosis and clinical progression of BC (P < 0.05). The molecular docking suggests that the active ingredients have a high affinity with hub gene. The proliferation results suggest that limonin inhibits MCF-7 cells in a dose- and timedependent manner. The results suggest that limonin may induce cell cycle arrest in the G2-M phase, allowing apoptosis to occur. Immunofluorescence and western blot results indicate that it exerts anti-tumour effects by down-regulating the expression of these proteins. This study identified potential biomarkers of LMD against BC. Notably, LMD can exert anti-tumour effects by inhibiting the proliferation of MCF-7 cells, down-regulating the expression levels of CCNB1 and AHURKA proteins, inducing cell cycle arrest in the G2-M phase, leading to apoptosis, and regulating the p53 signaling pathway. It is expected to provide certain guiding significance for the further exploration of potential markers and molecular regulation mechanisms for the diagnosis and prognosis of BC patients with LMD.