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

Abstract

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.

Keywords: H2O2 resistant cells; cell death; clinically relevant radioresistant cells; radiation therapy; reactive oxygen species.

Figure 1.

CRR cell lines are resistant to H₂O₂-induced cell death. Cells (5 × 10³) were incubated with H₂O₂ for 48 hours and a WST assay was performed according to the manufacturer’s protocol. A, H₂O₂ sensitivity of parental HepG2 and CRR HepG2-8960-R cells. B, H₂O₂ sensitivity of parental HepG2 and CRR HepG2-R cells. C, H₂O₂ sensitivity of parental SAS and CRR SAS-R cells. D, H₂O₂ sensitivity of parental HeLa and CRR HeLa-R cells. The data represent the mean ± SD from 3 independent experiments and are presented relative to the control parental cell lines. *: p < 0.05 using Student’s t test (vs. Parental cells).

Figure 2.

Generation of H₂O₂-resistant cell lines. The sensitivity of cells to H₂O₂-induced cell death was determined by WST assays. The H₂O₂-resistant cell lines are referred to as their cell line name followed by H₂O₂. The cells (5 × 10³) were incubated with H₂O₂ for 48 hours and a WST assay was performed according to the manufacturer’s protocol. A, H₂O₂ sensitivity of parental HepG2 and HepG2-H₂O₂ cells. B, H₂O₂ sensitivity of parental SAS and SAS-H₂O₂ cells. C, H₂O₂ sensitivity of parental HeLa and HeLa-H₂O₂ cells. The data represent the mean ± SD of 3 independent experiments and are presented relative to the control parental cell lines. *: p < 0.05 using Student’s t test (vs. Parental cells).

Figure 3.

H₂O₂-resistant cell lines are not resistant to X-ray-induced cell death. A, X-ray sensitivity of HepG2, CRR HepG2-8960-R, and HepG2-H₂O₂ cells. B, X-ray sensitivity of SAS, CRR SAS-R, and SAS-H₂O₂ cells. C, X-ray sensitivity of HeLa, CRR HeLa-R, and HeLa-H₂O₂ cells. The data represent the mean ± SD of 3 independent experiments and are presented relative to the control parental cell lines. *: p < 0.05 using Dunnett’s test (vs. Parental cells).

Figure 4.

Exposure to fractionated X-rays induced H₂O₂ resistance in cancer cells. Cells (5 × 10³) were incubated with H₂O₂ for 48 hours and a WST assay was performed according to the manufacturer’s protocol. A, H₂O₂ sensitivities of parental HepG2 cell, 0.5 Gy/day × 10 of fractionated X-rays pretreated HepG2 (HepG2-0.5) cell, 0.5 Gy/day × 10 plus 1 Gy/day × 10 of fractionated X-ray pretreated HepG2 (HepG2-0.5-1) cell and CRR HepG2-8960-R cell. B, H₂O₂ sensitivities of parental SAS, 0.5 Gy/day × 10 of fractionated X-rays pretreated SAS (SAS-0.5) cell, 0.5 Gy/day × 10 plus 1 Gy/day × 10 of fractionated X-ray pretreated SAS (SAS-0.5-1) cell and CRR SAS-R cells. C, H₂O₂ sensitivity of parental HeLa, 0.5 Gy/day × 10 of fractionated X-rays pretreated HeLa (HeLa-0.5) cell and CRR HeLa-R cell. The data represent the mean ± SD of 3 independent experiments and are presented relative to the control parental cell lines. *: p < 0.05 using Dunnett’s test (vs. Parental cells).

Figure 5.

The H₂O₂ resistance phenotype of CRR cell lines was irreversible. Cells (5 × 10³) were incubated with H₂O₂ for 48 hours and a WST assay was performed according to the manufacturer’s protocol. A, H₂O₂ sensitivity of parental HepG2, CRR HepG2-8960-R, and HepG2-8960-R-NoIR cells. B, H₂O₂ sensitivity of parental HepG2, CRR HepG2-R, and HepG2-R-NoIR cells. C, H₂O₂ sensitivity of parental SAS, CRR SAS-R, and SAS-R-NoIR cells. D, H₂O₂ sensitivity of parental HeLa, CRR HeLa-R, and HeLa-R-NoIR cells. The data represent the mean ± SD of 3 independent experiments and are presented relative to the control parental cell lines. *: p < 0.05 using Dunnett’s test (vs. Parental cells).

Figure 6.

Exposure of CRR cells to X-rays enhanced their resistance to H₂O₂-mediated cell death. Following incubation with H₂O₂ for 1-h, cells (5 × 10³) was irradiated with 10 Gy of X-rays and incubated extra 48 hours. WST assays were then performed according to the manufacturer’s protocol. A, H₂O₂ sensitivity of parental HepG2 and HepG2 irradiated cells. B, H₂O₂ sensitivity of CRR HepG2-8960-R and CRR HepG2-8960-R irradiated cells. C, H₂O₂ sensitivity of HepG2-R and HepG2-R irradiated cells. D, H₂O₂ sensitivity of Parental SAS and SAS irradiated cells. E, H₂O₂ sensitivity of CRR SAS-R and SAS-R irradiated cells. F, H₂O₂ sensitivity of parental HeLa and HeLa irradiated cells. G, H₂O₂ sensitivity of CRR HeLa-R and HeLa-R irradiated cells. The data represent the mean ± SD of 3 independent experiments and are presented relative to the control parental or CRR cell lines. *: p < 0.05 using Student’s t test (vs. no irradiated cells).

Figure 7.

The kinetics of intracellular ROS production in H₂O₂-treated parental and CRR cells. Cells were treated with or without 100 μM of H₂O₂ for 3 or 24 hours and the intracellular ROS levels were determined using the CellROX™ Green Reagent according to the manufacturer’s protocol.