Abstract
Objective: To determine the biological effectiveness of iodine 125 (125I) radioactive seeds continuous low dose rate radiation on the human esophageal cancer cell line KYSE150 compare to single dose radiation and explore its potential cellular mechanisms.Method: Three groups of KYSE150 cells were explored: control group, single dose irradiation (SDR) and 125I seeds continuous low dose rate irradiation group (125I-CLDR). Dose-survival curves was obtained by colony formation assay. MTT proliferation assay was used to measure KYSE150 cell vibility. KYSE150 cell apoptosis was analysis by Annexin V-FITC/PI staining, meanwhile, cell cycle analysis was performed by flow cytometry. Bim, Bcl-2, caspase-3 and cleaved caspase-3 protein expression were measured by western blotting to vertify the apoptosis level, as well as CyclinB1 protein expression which values the fuction of G2/M check point.Cell morphology changes were observated under phase contrast microscope. Endoplasmic reticulum stress (ER stress) was measured by GPR78/Bip1 and PERK changes in gene expression, which was detected by Real-time PCR. Reactive oxygen species(ROS) changes and mitochondria were meaured by flow cytometry. ATP detection kit was used to measured ATP level afer two modes of irradiation. DNA-Pks, Ku70 and Ku80 expression were measured by western blotting to represent the damage of DNA damage and repair capabilities. Acridine orange (AO) staining was used to detect level of autophagy and quantitative was measured by flow cytometry. LC3-II, ATG5, Beclin1, p-Akt, p-mTOR, mTOR and p-S6 proteins expression were detectd by western blotting.Results: KYSE150 cells were more radiosensitive to 125I-CLDR than SDR. Two modes of irradiation could both inhibit the proliferation vability of KYSE150 cells, while the inhibition effects in 125I-CLDR was significantly stronger compared with SDR. Compared with SDR, 125I-CLDR showed more proportions of the early and late apoptosis rate as well as cells at G2/M phase. Apoptosis related proteins, such as Bim, caspase-3 and cleaved caspase-3, were elevated, while CyclinB1 expression was decreased in 125I-CLDR group. Cells became bigger and grainy in 125I-CLDR group, meanwhile, ROS levels and ER stress signal was elevated except ATP concentration. Cells stained by AO was anaysised by flow cytometry, results showed that red: green ratio in 125I-CLDR group increased as well as LC3 and ATG5 protein expression detected by western blotting. p-Akt, p-mTOR and p-S6 protein expression were decreased to some extent in 125I-CLDR group cells, while total mTOR protein showed no significant changes.Conclusion: Our results confirmed that 125I-CLDR could strong inhibite KYSE150 cancer cells. Furthermore, we revealed that cell cycle arrest and apoptosis were not the only fate after 125I-CLDR, autophagy might be another important choice which mTOR pathway might be involed in. These findings can support the application of esophageal stent loaded with 125I seeds in clinic.
Relative Biological Effectiveness and Cell-Killing Efficacy of Continuous Low-Dose-Rate 125I Seeds on Prostate Carcinoma Cells In Vitro