Salinomycin Affect Ridiosensitivity Of The Nasopharyngeal Carcinoma Stem Cells

ObjectiverTo investigate the salinomycin affects radiosensitivity in nasopharyngeal carcinoma stem cells.Methods:Part 1:CNE-2 cells were cultured in SFM, and their micro-spheres morphology were observed by microscope.Then differentiation ability were observed by join SSM. Transwell chamber experiments,flow cytometry,colony formation assay were used to detect their invasiveness in vitro,CD133 expression proportion and cell proliferation in CNE-2 and CNE-2-CSC.Part 2:CNE-2-CSC cells culture in serum-free culture.Then MTT assay salinomycin dose response and different radiation doses CNE-2-CSC cell’s proliferation.MTS staining calculate the cell inhibition rate.Clonogenic assay test radiosensitizing effect of salinomycin on CNE-2-CSC cells.RT-PCR and Western Blot method detect Rad51,Ku70, Ku80 gene and protein expression level.Results:Part 1:In SFM,CNE-2 microscopic spherical cells grown in suspension with a strong refraction.After join SSM,CNE-2 microscopic spherical cells differentiation into similar CNE-2.CNE-2-CSC compared with CNE-2 cells had a strong invasiveness,(142±7)/HP vs (63±8)/HP,P<0.01.The positive expression rate of CD 133 in CNE-2-CSC is (98.29±1.28)%,which in higher than CNE-2 (1.58±0.28)%.The difference was statistically significant.Cloning forming test showed CNE-2-CSC has stronger ability of self-renewal.Part 2:MTT assay of salinomycin IC50= 3.026μM, SF2μM= 62.4± 3.3%,6MV-X-ray LD50= 8.226Gy, SF6Gy= 75.7±1.89%.The treatment with salinomycin and 6MV-X group inhibition rate obviously higher than the other tow group, there were significantly difference(P<0.05).The results of clone formation assay illustrated that salinomycin reduces colony forming efficiency of CNE-2-CSC cell after exposure.The indicators that represent radio sensitivity was calculated by the survival curve,which showed that salinomycin enhanced radiosensitivity.According to single-hit multi-target model,radiation enhancement ratio(SER) was 1.345.There were remarkable difference of Rad51,Ku70 and Ku80mRNA expression level between experimental group and the control group cells(P< 0.05).Experimental group Rad51, Ku70 and Ku80 protein expression levels evidently lower than the control group, the difference was statistically apparent (P<0.05).Conclusion:Serum-free medium culturing can be successfully cultured stem cells of CNE-2. CNE-2-CSC has a strong invasiveness, proliferation and a high expression of CD 133.Salinomycin can significantly inhibit Rad51, Ku70 and Ku80 expression in nasopharyngeal carcinoma cells, reduce the cell survival fraction, improve radiation sensitivity and clinical curative effect, for clinical guiding significance, is worth clinical promotion.