The Effects of Hydrogen Peroxide and/or Radiation on the Survival of Clinically Relevant Radioresistant Cells

Background: Radiation therapy is a highly cost-effective treatment for cancer, but the existence of radio-resistant cells remains the most critical obstacle in radiotherapy. We have been established clinically relevant radioresistant (CRR) cell lines by exposure to a stepwise increase of fractionated X-rays. We are trying to overcome the radio-resistance by analyzing the properties of these cells. In this study, we tried to evaluate the effects of hydrogen peroxide (H₂O₂) on the CRR cells because this can evaluate the efficacy of Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas (KORTUC) that treats H₂O₂ before irradiation. We also established H₂O₂-resistant cells to compare the radiation and H₂O₂ resistant phenotype. Materials and Methods: We used human cancer cell lines derived from hepatoblastoma (HepG2), oral squamous cell carcinoma (SAS), and cervical cancer (HeLa). We established HepG2, SAS, and HeLa CRR cells and HepG2, SAS, and HeLa H₂O₂-resistant cells. To evaluate their sensitivity to radiation or H₂O₂, high-density survival assay, or WST assay was performed. CellROX^TM was used to detect intracellular Reactive Oxygen Species (ROS). Results: CRR cells were resistant to H₂O₂-induced cell death but H₂O₂-resistant cells were not resistant to irradiation. This phenotype of CRR cells was irreversible. The intracellular ROS was increased in parental cells after H₂O₂ treatment for 3 h, but in CRR cells, no significant increase was observed. Conclusion: Fractionated X-ray exposure induces H₂O₂ resistance in CRR cells. Therefore, it is necessary to carry out cancer therapy such as KORTUC with the presence of these resistant cells in mind, and as the next stage, it would be necessary to investigate the appearance rate of these cells immediately and take countermeasures.