Quercetin induces molecular hallmarks of immunogenic cell death and cGAS-STING pathway activation via mitochondrial DNA release in hepatocellular carcinoma cells.

Immunotherapy for hepatocellular carcinoma (HCC) is constrained by the immunosuppressive tumor microenvironment. Inducing immunogenic cell death (ICD) and activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway are promising strategies to reinvigorate anticancer immunity. Quercetin (QUR) exerts extensive anti-HCC effects, yet whether it can provoke ICD and STING pathway activation remains unknown. Human HCC cell lines SMMC‑7721 and Huh‑7 were treated with various concentrations of QUR. Cell proliferation, colony formation, migration, invasion, and apoptosis were assessed by CCK‑8, EdU, wound‑healing, Transwell, and YO‑PRO‑1/PI assays. The release of damage-associated molecular patterns (DAMPs), which represent characteristic hallmarks of ICD, was systematically evaluated: immunofluorescence for cell surface calreticulin (CRT) exposure, ELISA for extracellular adenosine triphosphate (ATP) levels, quantitative real-time PCR (qPCR) for cytosolic mitochondrial DNA (mtDNA) release, and western blotting for the protein expression of heat shock protein 70 (HSP70), HSP90, and high mobility group box 1 (HMGB1). cGAS-STING signaling activation was assessed by measuring the protein levels of cGAS and STING, along with the phosphorylation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and p65. QUR dose‑dependently inhibited proliferation, colony formation, migration, and invasion, while promoting apoptosis in both HCC lines. QUR triggered the release of DAMPs, as shown by increased cell surface CRT exposure, extracellular ATP release, cytosolic mtDNA leakage, and upregulation of HSP70, HSP90, and HMGB1. Concurrently, QUR activated the cGAS‑STING pathway, evidenced by elevated expression of cGAS and STING, along with enhanced phosphorylation of TBK1, IRF3, and p65. Our findings suggest that QUR concurrently elicits molecular hallmarks of ICD and activates the cGAS‑STING pathway through mtDNA release in HCC cells, which provides a preliminary mechanistic basis for exploring QUR as an immunomodulatory agent for HCC.