Multiomics and experimental validation reveal theophylline's mechanism targeting IL1A/ACTB/TLR4 and identify synergistic drugs in hepatocellular carcinoma.

This study aimed to investigate the mechanism by which theophylline influences hepatocellular carcinoma (HCC) through the regulation of core targets and to identify its potential synergistic drugs. Integrated network pharmacology, multiomics data (transcriptomics, single-cell, and spatial transcriptomics), and multiple machine learning algorithms (a total of 113 diagnostic models combined with SHapley Additive exPlanations, least absolute shrinkage and selection operator, and artificial neural network analyses) were used to screen core targets linking theophylline, gut microbiota, and HCC. Enrichment analyses (Disease Ontology, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes) were conducted to elucidate their biological functions. Molecular docking and molecular dynamics simulations were used to evaluate the binding patterns and stability between theophylline (and potential synergistic drugs) and the core targets. Databases including The University of ALabama at Birmingham CANcer data analysis Portal, Gene Expression Profiling Interactive Analysis, Comparative Toxicogenomics Database, and gutMGene were used to analyze the clinical relevance of core targets, their regulatory roles in the immune microenvironment, and their connections within the "theophylline-target-HCC chemical-gut microbiota" network. Finally, in vitro cell experiments (proliferation, migration, invasion, quantitative reverse transcription polymerase chain reaction, and Western blot) and in vivo xenograft models were used to validate the findings. Seventeen shared targets were screened, and 3 core targets, interleukin 1α, actin β, and toll like receptor 4, were further identified. Molecular docking and dynamics simulations demonstrated that theophylline could stably bind to these core targets. Multiple drugs including chlorogenic acid (PubChem CID: 1794427), losartan (PubChem CID: 3961), and estrone sulfate (PubChem CID: 3001028), were found to potentially exhibit synergistic effects with theophylline. The expression of core targets was significantly associated with clinical stage, prognosis, immune cell infiltration (eg, monocytes and macrophages), and immune checkpoints (including programmed death-ligand 1 and cytotoxic T-lymphocyte associated protein 4) in patients with HCC. Single-cell and spatial transcriptomic analyses revealed heterogeneous expression of core targets within the tumor microenvironment. Experimental validation confirmed that theophylline significantly inhibited HCC cell proliferation, migration, and invasion, downregulated core target expression, and effectively suppressed tumor growth in vivo. Theophylline may exert anti-HCC effects by regulating the core targets interleukin 1α, actin β, and toll like receptor 4, thereby influencing the tumor immune microenvironment, malignant biological behaviors of cells, and the gut microbiota-liver axis. This study provides a theoretical foundation for theophylline as a potential therapeutic or adjuvant agent for HCC and suggests directions for its combined application with specific drugs. SIGNIFICANCE STATEMENT: The research uncovers a new target and mechanism for the classic drug theophylline in treating hepatocellular carcinoma. This finding provides a key scientific basis for repurposing the drug. Furthermore, a drug combination discovered through multiomics analysis and laboratory tests offers a direct and practical new path for developing new combination therapies for hepatocellular carcinoma in the clinic.