| Objective:(1)To investigate the effect of radioactive protective agent EGCG on the outcome of radiotherapy for stage Ⅲ breast cancer.(2)To investigate the effect of EGCG on breast cancer cell line 4T1 after ionizing irradiation in vivo.(3)To verify the effect of topical EGCG on radiotherapy of breast cancer when it is used as a protective agent for radiation skin injury.Methods:(1)patients with stage III breast cancer who received EGCG and radiotherapy were selected from the phase II clinical trial(Clinical Trials.gov,NCT02580279).Patients with staging and age matching were randomly selected from the medical database of our hospital as the control group.The survival analysis and radiation skin injury of the two groups were analyzed.(2)4T1 was selected as the experimental cell line.CCk8 detected cell survival after EGCG pretreatment,clone formation assay revealed clone formation ability,flow cytometry revealed apoptosis rate,and WB revealed the expression level of apoptotic protein.Immunofluorescence revealed DNA damage,while a specific ROS fluorescence probe identified the ROS level.(3)Balb/c mice were selected as the experimental subjects.Firstly,the tumor-negative mice model was established by subcutaneous planting tumor,and then the acute radiation skin injury model was established by radiotherapy.At the same time,EGCG solution or normal saline was used locally.The tumor volume of mice was monitored with Vernier caliper and the tumor was intuitively understood by in vivo imaging.Two weeks after radiotherapy,the skin and tumor-free mice of each group were taken for HE staining and immunohistochemistry to evaluate the skin and tumor tissue.Results:(1)We finally enrolled 43 patients with EGCG and 43 matched controls.No substantial divergence in the original demographic or clinical features was observed between the two sets.The mean radiation dermatitis index(RIDI)in EGCG group was significantly lower than that in placebo group(2.56±1.14 vs 3.36 ±1.16).Repeated measurement of ANOVA showed that there was a significant difference in RTOG score between the two groups during radiotherapy(Fang 9.611).There was no significant difference in overall survival rate(OS),disease-free survival rate(DFS),local recurrence(LRF)and distant metastasis(DMF)between the two groups.(2)Cell experiments in vitro show that radiotherapy can reduce cell survival rate,reduce cell clone formation ability,induce cell apoptosis,increase cell ROS level,and lead to cell DNA damage.EGCG could potentially diminish the endurance of cells caused by ionizing radiation,impede the production of cell clones,and augment the apoptosis rate of tumor cells brought about by ionizing radiation,in comparison to the radiotherapy group.Compared with the radiotherapy group,EGCG could further increase the level of apoptotic protein BAX,cleaved caspase3 induced by ionizing radiation,increase the increase of ROS induced by ionizing radiation,and increase the damage of DNA induced by ionizing radiation.(3)from the tumor growth curve of mice,radiotherapy significantly inhibited t-he tumor growth of mice.However,compared with the radiotherapy group,local topical application of EGCG had no significant effect on tumor growth in mice.In vivo imaging can directly see the inhibitory effect of radiotherapy on tumor growth in mice treated with radiotherapy alone and EGCG combined with radiotherapy.Conclusion: EGCG as a protective agent for radiation-induced skin injury does not hinder the efficacy of radiotherapy for breast cancer. |
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