Unveiling the Novel Anti - Tumor Potential of Digitonin, a Steroidal Saponin, in Gastric Cancer: A Network Pharmacology and Experimental Validation Study

Dongdong Lu,1,2 Leijie Huang,1 Chunyan Weng2 1Department of Gastroenterology, Ningbo No. 2 Hospital, Ningbo, Zhejiang Province, 315000, People’s Republic of China; 2Department of Gastroenterology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, Zhejiang Province, 310000, People’s Republic of ChinaCorrespondence: Chunyan Weng, Email [email protected]: Gastric cancer (GC) remains a leading cause of cancer-related mortality, with limited effective treatment options for advanced stages. As a steroidal saponin with documented anti-neoplastic properties in multiple cancers, digitonin’s mode of action in GC pathogenesis has yet to be fully elucidated. This research focused on exploring the potential of Digitonin in GC treatment using a combination of network pharmacology and experimental validation.Methods: The inhibitory effects of Digitonin on the proliferation, invasion, and migration of gastric cancer cells were evaluated using CCK-8, colony formation, wound healing, and transwell assays. Key targets of Digitonin were identified through network pharmacology. Molecular docking and various experiments, including Western blot, immunofluorescence, and a subcutaneous xenograft model, were used for validation.Results: Digitonin exhibited stronger cytotoxicity against GC cells and significantly inhibited GC cell proliferation, migration, and invasion. Network pharmacology analysis revealed that the core targets of Digitonin are involved in key cancer-related signaling pathways, including HIF-1α, Ras, and PI3K-Akt pathways, with HSP90AA1 and NFKB1 identified as central targets. Further molecular docking, Western blotting, and immunofluorescence experiments confirmed that Digitonin significantly suppressed the expression of HSP90AA1 and inhibited the nuclear translocation of NFKB1, inducing cell apoptosis. Additionally, a subcutaneous xenograft model of GC further validated that Digitonin effectively inhibited tumor growth.Conclusion: Digitonin serves as a promising multi-target therapeutic agent for GC. This study underscores the potential of combining network pharmacology with traditional Chinese medicine to identify novel therapeutic targets and develop effective anti-cancer strategies. In addition, these findings suggest that digitonin could be a promising candidate for future clinical trials in GC treatment.Keywords: gastric cancer, digitonin, network pharmacology, HSP90AA1, NFKB1