Cell Death Induced by the Combination of Ephedra sinica Extract and Radiation in HNSCC is Positively Related to BAX and p-MLKL Expression

Background: Numerous studies have proven the efficacy and safety of natural products, and are wide-ly used as attractive cancer treatments. The investigation of effective natural products for improving cancer treatment is a promising strategy. Combination treatment with radiosensitizers and radiotherapy (RT) is consid-ered necessary for therapeutic improvement in head and neck squamous cell carcinoma(HNSCC). Objective: This study aims to investigate whether Ephedra sinica (ES) extract could induce selective cell death in cancer cells and serve as a radiosensitizer for HNSCC. Methods: HNSCC cells were pretreated with ES extract before radiation, and the radiosensitizing activity was assessed using a colony formation assay. Radiation-induced cell death was evaluated using an annexinV-FITC assay. Western blotting was performed to confirm cell death-related gene expression, including apoptosis and necrosis markers. Results: ES extract significantly inhibited HNSCC cell viability (FaDu and SNU1076), while having minimal effect on normal HaCaT cells. When HNSCC cells were irradiated with 2, 4, or 8 Gy and cultured with ES extract (25 μg/mL), they exhibited increased radiation sensitivity compared to non-treated cells. The combination of ES extract and radiation resulted in increased cell death compared to non-treated, ES-treated, or irradiated cells. The apoptosis marker BAX and necrosis marker p-MLKL expression levels were also elevated following the combination treatment. Conclusion: ES extract demonstrated significant cytotoxic potential in HNSCC cells without affecting normal cells. It enhanced the radiosensitivity of HNSCC cells by upregulating BAX and p-MLKL expression, leading to increased cell death. These results suggest ES extract exhibits a potential radiosensitizing capacity in HNSCC.