Identification of GDP as a small inhibitory molecule in HepG2 cells by non‑targeted metabolomics analysis

Identifying the mechanism by which lipid metabolism regulates cancer may offer a novel approach for therapeutic intervention. It has previously been identified that a lipid metabolism-related factor, namely fatty acid hydroxylase domain containing 2 (FAXDC2), is downregulated in various types of cancer, and inhibits the proliferation and migration of liver cancer cells through a mechanism associated with ERK. The liver is important for lipid metabolism, and FAXDC2 is involved in the synthesis of cholesterol and sphingomyelin. However, the functional mechanism by which FAXDC2 influences liver cancer cells through metabolic processes and ERK signaling remains unclear. Therefore, the present study induced the overexpression of FAXDC2 in HepG2 liver cancer cells and performed a metabolomics analysis. This identified guanosine diphosphate (GDP) as a significantly altered metabolite. Using AlphaFold3, a robust interaction was predicted between FAXDC2 and GDP, which lead to the hypothesis that GDP may mediate the inhibitory effects of FAXDC2 on liver cancer cells by directly modulating the functional properties of the cells, thereby influencing their behavior and progression. Cell Counting Kit-8 assays were used to study the impact of elevated GDP concentrations on HepG2 cell growth. The results revealed a gradual reduction in the viability of HepG2 cells as the GDP concentration increased. In addition, western blotting showed that GDP treatment was accompanied by a significant downregulation of cyclin dependent kinase 4 and cyclin D1 expression levels, and Transwell experiments revealed that GDP treatment significantly decreased the invasion of HepG2 cells. Treatment with GDP also significantly inhibited the expression of ERK. In summary, the present study is the first to indicate that GDP is a metabolic small molecule with inhibitory activity in cancer cells, which has previously been overlooked in tumor metabolic reprogramming. The study findings offer new insights and strategies for the diagnosis and treatment of liver cancer, and potentially other types of cancer.