Yajun Zhang,1,* Mingqin Zhou,2,* Liwen Zhu,3,* Lichan Chen,4 Haohua Zhang,3 Zhen Huang,5 Hongzhong Zhou3 

1Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China; 2Department of Critical Care Medicine, Cancer Hospital of Shantou University Medical College, Shantou, People’s Republic of China; 3Department of Laboratory Medicine, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Medical Innovation Technology Transformation Center of Shenzhen Second People’s Hospital, School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, People’s Republic of China; 4Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, People’s Republic of China; 5Department of Laboratory Medicine of Pingshan District Maternal and Child Healthcare Hospital, Shenzhen, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Zhen Huang, Email 635999667@qq.com Hongzhong Zhou, Email zhouhongzhong888@163.com

Purpose: This study aims to evaluate the therapeutic potential of tubeimoside I (TBMS1), a monomer compound extracted from the tubers of Chinese herb Bolbostemma paniculatum (Maxim). Franquet (Cucurbitaceae), in the treatment of liver cancer. Specifically, we sought to elucidate the underlying mechanisms through which TBMS1 exerts its anticancer effects.
Methods: The effects of TBMS1 on the viability, proliferation, and apoptosis of two liver cancer cell lines, MHCC97-H and SNU-449, were comprehensively assessed using Cell Counting Kit-8 (CCK-8), colony formation, 5-ethynyl-2′-deoxyuridine (EDU) assay, and flow cytometry assays. To uncover the molecular mechanisms, RNA sequencing was performed to identify the downstream targets of TBMS1. Additionally, we utilized network pharmacology to predict TBMS1 targets in liver cancer and employed Venn diagram analysis to integrate these predictions with our experimental findings. Pathway enrichment analysis was conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases to elucidate the biological processes involved. Furthermore, a subcutaneous xenograft tumor model was established to investigate the in vivo antitumor efficacy of TBMS1.
Results: In vitro experiments demonstrated that TBMS1 significantly enhanced cell apoptosis and inhibited the growth of liver cancer cells. Both network pharmacology predictions and RNA-seq analyses revealed that the downstream target genes of TBMS1 were highly enriched in the NF-κB signaling pathway. Notably, we observed a significant upregulation of TNFα-induced protein 3 (TNFAIP3) expression with increasing concentrations of TBMS1. In vivo studies further confirmed that TBMS1 treatment dramatically reduced the volume and weight of liver cancer tumors compared to controls.
Conclusion: Our study provides compelling evidence that TBMS1 suppresses liver cancer progression by inactivating the NF-κB pathway and regulating TNFAIP3 expression. These findings offer novel insights and a theoretical basis for the development of targeted therapies for liver cancer.

Keywords: tubeimoside-I, hepatocellular carcinoma, TNFAIP3, NF-κB, proliferation