Fufang Changtai Decoction Inhibites Colorectal Cancer Through Ferroptosis: Investigation of the Underlying Mechanism
Abstract
To investigate the underlying mechanisms of the effect of Fufang Changtai Decoction (FFCT) in inhibiting colorectal cancer (CRC) through the ferroptosis pathway using network pharmacology combined with experimental validation.
Methods
The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Swiss Target Prediction databases were employed for the systematic screening of potent active ingredients and therapeutic targets of FFCT. In addition, the identification of CRC-associated genes and ferroptosis-related genes (FRGs) was accomplished using the Gene Cards and FerrDb databases, respectively. Venn diagrams, coupled with Cytoscape software, facilitated the comprehensive analysis of key FRGs involved in FFCT's intervention in CRC by mapping the TCM compound-therapeutic target network. Transmission electron microscopy was used to examine the mitochondrial ultrastructure of SW480 and HCT116, 2 Human CRC cell lines, after treatment with FFCT-containing serum. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit. To assess the role of ferroptosis, ferroptosis inhibitor liproxstatin-1 (Lip-1) was co-administered with FFCT-containing serum. The effects on cancer cell viability and proliferation were evaluated using CCK-8 and colony formation assays. Key molecular targets involved in the regulatory effects of FFCT on the expression of FRGs were further analyzed using PCR Array and Western blot. The findings were then validated with human CRC tissue microarrays.
Results
A total of 103 active ingredients of FFCT, 739 therapeutic targets, 9101 disease-related genes, and 564 FRGs were identified. Venn diagram analysis identified 81 FRGs associated with FFCT intervention. Network analysis revealed that NQO1, TP53, and PTGS2 served as hub nodes in the regulatory network. Findings from the in vitro experiments showed that FFCT induced ferroptosis changes, including mitochondrial condensation, membrane thickening, and cristae reduction, in SW480 and HCT116 cells. FFCT treatment significantly increased intracellular ROS levels in a dose-dependent manner (P < 0.05) and reduced cancer cell viability and proliferative capacity (P < 0.01). These inhibitory effects were partially reversed by Lip-1, suggesting that FFCT's antitumor activity was closely associated with the ferroptosis pathway. PCR Array and Western blot analyses further confirmed that FFCT significantly downregulated NQO1 mRNA and protein expression in cancer cells (P < 0.001), which was consistent with network pharmacology predictions. Immunofluorescence analysis of clinical CRC tissue microarrays revealed that NQO1 expression was significantly higher in tumor tissues than in adjacent non-tumor tissues (P < 0.001).
Conclusion
FFCT may induce intracellular ferroptosis by downregulating the oncogenic gene NQO1, thereby exerting anti-CRC effects.
Keywords: Fufang Changtai Decoction, Colorectal cancer, Ferroptosis, Network pharmacology, Experimental validation
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