Abstract
BACKGROUND: Enhanced glycolytic levels in cancer cells are a common characteristic of many cancer types. Modulation of glycolytic metabolism is crucial for enhancing the efficacy of cancer therapy. The specific role of 6-methoxyflavone in regulating glycolytic metabolism in cancer cells remains unclear. This study aimed to elucidate the impact of 6-methoxyflavone on glycolytic metabolism in cervical cancer cells and its clinical relevance. METHODS: The tandem mass tag (TMT) proteomic analysis was used to identify significantly enriched biological processes and pathways in HeLa cells after treatment with 6-methoxyflavone. Additionally, the differential expression of glycolysis-related proteins was validated using parallel reaction monitoring (PRM) proteomics. Untargeted and targeted metabolomics analyses were used to identify differentially expressed glycolysis-related metabolites. Furthermore, alternative splicing, new transcripts, and domain analyses were used to detect the effects of 6-methoxyflavone on the structures of glycolysis-related genes and proteins. Subcellular localization, molecular docking, and non-covalent interaction analyses were used to detect the subcellular localization, affinity of 6-methoxyflavone for glycolysis-related proteins, and sites of non-covalent interactions. Clinical characteristics and immunological correlation analyses were used to elucidate the relationships between glycolysis-related genes and clinicopathological characteristics, survival, prognosis, and immune-related indicators of patients with cervical cancer. Finally, glycolysis stress tests and enzyme activity assays were used to verify the effect of 6-methoxyflavone on glycolysis in HeLa cells. RESULTS: TMT and PRM proteomics, as well as untargeted and targeted metabolomics results, showed that 6-methoxyflavone downregulated the expression levels of glycolysis-related proteins and metabolites in HeLa cells, and that the structures and functions of glycolysis-related genes and proteins in the cytoplasm underwent changes. 6-Methoxyflavone had a good affinity for nine glycolysis-related proteins, all of which had non-covalent interaction sites. Clinical characteristics and immune correlation analyses showed relationships between 6-methoxyflavone and five clinical characteristics, survival prognosis, and four immune-related indicators in patients with cervical cancer. After treatment with 6-methoxyflavone, the basal glycolytic level, maximum glycolytic capacity, and glycolytic reserve of HeLa cells were downregulated. Additionally, 6-methoxyflavone inhibited the activity of pyruvate kinase. CONCLUSION: 6-Methoxyflavone inhibited energy metabolism in HeLa cells through the glycolysis pathway. 6-Methoxyflavone may be related to five clinical characteristics, prognosis, tumor microenvironment, immune cells, immune checkpoints, and immunotherapy efficacy in patients with cervical cancer.