Assessment of gut microbiota dynamics and probiotic impact on HPV-associated head and neck cancer using 16S rRNA sequencing and cytokine profiling.

Introduction: Human papillomavirus (HPV) is a major etiological factor in head and neck cancers, primarily through the oncogenic activities of viral proteins E6 and E7, which disrupt the p53 and retinoblastoma (Rb) tumor suppressor pathways. This disruption promotes uncontrolled cell proliferation, immune dysregulation, chronic inflammation, and malignant transformation. Although probiotics have emerged as promising modulators of the microbiome and host immune responses, their mechanistic role in HPV-associated head and neck cancer remains insufficiently understood. Therefore, this study investigated the therapeutic potential of selected probiotic strains and their metabolic derivatives against HPV-positive head and neck cancer using integrated experimental and computational approaches. Methods: Three probiotic strains Lactobacillus rhamnosus, Lactobacillus plantarum, and Bifidobacterium longum were evaluated as live cultures, heat-killed preparations, extracellular vesicles, and metabolite extracts in HPV-positive head and neck cancer cell models. Microbial community alterations were analyzed using 16S rRNA sequencing, while functional pathway prediction was performed through PICRUSt2 and KEGG enrichment analyses. Cellular responses were assessed using MTT assays for cell viability, BrdU incorporation for proliferation, and Annexin V-FITC/PI staining with caspase activity assays for apoptosis. Cytokine profiling was conducted to evaluate immune modulation. Network-based systems biology analyses were performed using Cytoscape and protein-protein interaction mapping. Molecular docking simulations using AutoDock Vina examined the binding interactions of probiotic-derived metabolites with HPV16 E6 and E7 oncoproteins. Results and Discussion: Probiotic treatments significantly reshaped the microbial community by enriching short-chain fatty acid (SCFA)-producing beneficial taxa while reducing pathogenic Proteobacteria. Functional analyses indicated enhanced SCFA biosynthesis, IL-10-mediated immune regulation, oxidative stress defense, and epithelial barrier maintenance pathways. In vitro experiments demonstrated significantly reduced cancer cell viability and proliferation together with increased apoptosis, supported by elevated caspase-3 and caspase-9 activities. Increased production of IL-10 and TGF-β further confirmed immunoregulatory effects. Molecular docking revealed strong binding affinities of probiotic-derived metabolites, particularly butyrate and bacteriocins, toward HPV16 E6 and E7 proteins, suggesting potential inhibition of viral oncogenic activity. Collectively, these findings demonstrate that probiotics exert microbiome-associated, immunomodulatory, and anti-oncogenic effects in HPV-positive head and neck cancer models, supporting their potential as promising adjunctive therapeutic agents for HPV-related malignancies.