Exploration of the Role and Mechanism of Proton Beams In Radiotherapy-Resistant Cells
Abstract
Purpose
Radiotherapy is a standard treatment for lung cancer, yet 40%–50% of patients develop local failure or recurrence, largely due to radioresistance. Proton beams, characterized by the Bragg peak and higher relative biological effectiveness, may enhance tumor killing, especially in resistant cells. This study examined whether protons exert superior cytotoxicity on radiotherapy‑resistant non-small cell lung cancer A549 cells and explored the underlying mechanisms.
Methods
A549 cells were irradiated with 2–10 Gy X‑rays to determine a sublethal dose; 6 Gy was identified as suitable for stable passaging. Radioresistant lines were established by ≥5 rounds of 6 Gy: X‑ray‑resistant A549X and proton‑resistant A549R (protons: 110 MeV, cells at the middle of the SOBP). Clonogenic assays were performed under photon and proton irradiation. Under X‑rays, survival fractions of A549X vs A549R were 0.437 vs 0.626 (2 Gy), 0.137 vs 0.276 (4 Gy), 0.084 vs 0.109 (6 Gy), 0.035 vs 0.048 (8 Gy), and 0.010 vs 0.013 (10 Gy). Under proton irradiation, survival fractions were 0.144 vs 0.211 (2 GyE), 0.204 vs 0.228 (4 GyE), 0.071 vs 0.118 (6 GyE), 0.027 vs 0.053 (8 GyE), and 0.005 vs 0.020 (10 GyE), indicating stronger proton killing of photon‑resistant cells.
Results
RNA‑seq of A549, A549X, and A549R (12 samples) identified 35,921 expressed genes. Compared with parental cells, photon‑ and proton‑resistant cells shared 2,667 differential genes, with 54.6% overlap. Between A549X and A549R, 221 genes were differentially expressed (61 upregulated, 160 downregulated). GO, KEGG, and Reactome analyses revealed distinct enrichment in inflammatory (e.g., IL‑17, TNF) and immune pathways (e.g., NK cell–mediated cytotoxicity).
Conclusion
In conclusion, proton and photon irradiation generate radioresistant A549 cells with distinct transcriptomic profiles. Protons more effectively kill photon‑resistant cells, potentially via modulation of inflammatory and immune responses.