Berberine Enhances Radiosensitivity of Head and Neck Squamous Cell Carcinoma Concurrent with the Inhibition of DNA Repair, Stemness and Tumor Growth

Background/Objectives: The emergence of radioresistance is a major challenge in cancer treatment. Herein, we evaluated the radiosensitizing effects and associated molecular mechanisms of a small molecule, berberine, in HNSCC. Methods: HNSCC cells were treated with berberine, ionizing radiation (IR), and their combination. Radiosensitivity was assessed using methods for clonogenic survival, proliferation, cell death, apoptosis, cell cycle distribution, DNA damage, and associated mechanisms. Spheroid models were employed to examine tumor growth and stemness-related markers. Therapeutic efficacy was further evaluated using the syngeneic MOC2 tumor syngraft mouse model. Results: The combination of berberine and IR significantly decreased the colony-forming ability, cell proliferation, and survival in UM-SCC-22B and MOC2 cells. Correspondingly, a decrease in the pro-survival signaling, EGFR, ERK1/2, mTOR, and STAT-3 was noted. The combination treatment increased sub-G1 cell population and apoptotic cell death with enhanced DNA damage, which was also associated with downregulation of DNA repair proteins. Spheroids showed disintegration and altered morphology in the combination treatment. This combination also inhibited Wnt/β-catenin signaling and reduced the expression of genes associated with stemness-related pluripotency. Berberine further enhanced DNA damage and reduced stemness in EGFR-knockdown cells. The berberine-IR combination not only inhibited the growth of syngeneic MOC2 tumors but also mitigated the toxicity associated with IR. The in vitro findings of reduced expression of stemness and DNA damage repair genes were also observed in tumors. Conclusions: Our findings indicated that berberine enhances the radiation response of HNSCC through perturbation of pro-survival signaling, DDR signaling and reduction of stemness-associated features, thereby enhancing the therapeutic efficacy.