Investigation of 8-O-acetylharpagide induced G2/M arrest and radiosensitization effects on hypopharyngeal cancer

[Purpose] AT-1 (8-O-acetylharpagide) is a natural compound purified from the endemic Taiwanese plant Ajuga taiwanensis and has been reported to possess anti-aging, anti-inflammatory, and anti-cancer properties, such as skin, liver and breast, but its potential role in Hypopharyngeal squamous cell carcinoma (HPSCC) remains unclear. This study aimed to systematically characterize the cellular responses to AT-1 in human hypopharyngeal carcinoma cells (FaDu) versus normal human periodontal ligament fibroblasts (PDL) and to evaluate its selective cytotoxic and radiosensitizing potential. [Methods] Cell viability was assessed using the MTT assay. Flow cytometry was performed to analyze cell-cycle distribution and apoptosis. DNA damage was evaluated by immunofluorescence staining and comet assay. Western blotting was used to detect changes in cell cycle- and apoptosis-related proteins. Radiation sensitivity was examined following AT-1 pretreatment and subsequent X-ray exposure. [Results] AT-1 selectively reduced the viability of FaDu cells while exerting minimal cytotoxicity in PDL cells and induced pronounced G2/M phase arrest in FaDu, accompanied by abnormal upregulation of Cyclin B1 and modulation of cell cycle regulators. Apoptosis analysis revealed that AT-1 triggered cell death through downregulation of pro-survival proteins including AKT, p-AKT, and Bcl-2. Intracellular reactive oxygen species levels did not significantly increase, suggesting that oxidative stress was not directly responsible for AT-1-induced cytotoxicity. Immunofluorescence and comet assays confirmed the presence of DNA double-strand breaks in AT-1-treated FaDu cells, which may underlie the observed G2/M arrest and apoptosis. Furthermore, AT-1 pretreatment enhanced the radiosensitivity of FaDu cells, whereas PDL cells remained relatively resistant to AT-1 alone and to the combination treatment. AT-1 inhibits the growth of hypopharyngeal carcinoma cells by perturbing the cell cycle, inducing DNA damage, and promoting apoptosis, and it acts as a selective radiosensitizer in this model. [Conclusions] These data demonstrate that AT-1 (8-O-acetylharpagide) induces G2/M arrest and selectively radiosensitizes hypopharyngeal cancer cells while sparing normal cells. AT-1 may represent a promising low-toxicity radiosensitizer candidate for head and neck cancer, and further in vivo and mechanistic studies are warranted to define its impact on DNA damage response.