Human Urine Sediment Induces Apoptosis in Esophageal Squamous Cell Carcinoma through the Nonanedioic Acid–BAK1 Axis: Network Pharmacology, Molecular Docking, and In Vitro Validation

Esophageal squamous cell carcinoma (ESCC) remains refractory to chemoradiotherapy and relapses frequently. Human urine sediment, a traditional medicine known as Ren Zhong Bai, has historical indications for throat ulceration, yet its anticancer mechanism is undefined. Integrated mass spectrometry, network pharmacology and molecular docking were applied to link HUS constituents with differentially expressed genes from The Cancer Genome Atlas. Immune infiltration and drug sensitivity were explored bioinformatically. In vitro efficacy was assessed in KYSE‑30 and KYSE‑150 cells by Cell Counting Kit‑8 proliferation assay and Annexin V flow cytometry. Four hundred forty‑six HUS target genes overlapped with tumor gene dysregulation and were enriched for apoptotic and PI3K–Akt pathways. Serum metabolomics identified 15 absorbed compounds, and Nonanedioic acid uniquely docked with high affinity to lysine-87 of the pro‑apoptotic protein BAK1. Tumors showed higher BAK1 expression than normal tissue and worse differentiation with elevated expression. High BAK1 correlated with resting natural killer cell infiltration, while low BAK1 predicted greater sensitivity to multiple cytotoxic and targeted agents. HUS serum significantly reduced proliferation and increased apoptosis in both ESCC lines, whereas blank serum had no effect. These findings pave the way for further research to validate that HUS exerts antiproliferative activity in ESCC through a Nonanedioic‑acid–BAK1 axis that activates mitochondrial apoptosis and reshapes the immune microenvironment. BAK1 emerges as a dual biomarker for HUS responsiveness and drug stratification, providing a molecular rationale to repurpose a traditional remedy as an adjunct to current therapy.