Effect Of Aresenic Trioxide's Increasing Radiosensitivity On Human Cervical Carcinoma Hela Cell Line

Objective:Observe whether As₂O₃ can kill the human cervical carcinoma Hela cell by way of increasing radiosensitity.Then explore it's mechanism to provide some experimental references to the treatment of cervical carcinoma using As₂O₃ combined with radiation therapy.Methods:MTT assay was used to analysis the cell cytotoxics of Hela cell line.Cell growth inhibition rate=(1-OD values_{experimental groups}/ OD value_{control group})×100%.The experiments were repeated 3 times.Cell growth inhibiting curve was drew by excel software with the horizontal axis and longitudinal axis representing concentration of As₂O₃ and cell growth inhibition ratio respectively.The half inhibitive dosage(ID₅₀) was calculated by the software for ID₅₀calculation.Clone forming assay was carried out to take count of survival fraction(i.e. cell survival rate)on the 20%ID₅₀concentration of As₂O₃ combined with irradiation and colonies with cells exceeded 50 were calculated.Colony forming efficiency (CFE)was the number of colonies with above 50 cells/the number inoculated.The cell survival rate=(CFE_{experimental groups}/ CFE_{control group})×100%.The experiments were repeated 3 times.The cell suvrival cuvres were detemrined by cloning assay for evaluating the cell killing effets in the groups of irradiation plus As₂O₃ and the groups of irradiation alone.Survival curve was fitted by SPSS13.0 using multi-target single-hit model and its X-axis and Y-axis represented radiation dose and survival fraction respectively.Then the sensitization enchancement ratio(SER)was calculated.The cell cycle was determined by Flow cytometry.The experiments were divided four groups:control group,20%ID₅₀of As₂O₃ group,radiation therapy group and radiation therapy plus 20%ID₅₀of As₂O₃ group.Three parallel samples were installed for each group.1×10⁵ cells were inoculated into culture flask of 25 cm².Collecting the treated cells of each group,then fixed and stained.At last,cell cycle was detected by flow cytometry and the results were analyzed by the multicycle software.Immunocytochemistry and western blotting were applied to detect the expression of p21^waf1/cip1and Bcl-2 of control group,20%ID₅₀of As₂O₃ group,radiation therapy group and radiation therapy plus 20%ID₅₀of As₂O₃ group.All the performance was carried out according to the kit instruction.Results:1.The growth inhibiting of As₂O₃ to Hela cells of Human cervical Carcinoma was enhanced with the increase of As₂O₃ concentration.After treated with As₂O₃ for 48h, the ID₅₀was10.86μmol/L.2.Both the average deadly dosage(D₀)and quasi-domain dose(D_q)of Hela cells treated with As₂O₃ were less than that of sole radiation therapy group(D₀:2.49 Gy vs 3.45 Gy,D_q:1.03 Gy vs 1.89 Gy).The sensitization enchancement ratio(SER)was 1.39.3.The percentage of G₂/M period cells increased after treated with As₂O₃ or radiation or As₂O₃ combining with radiation.The mean value of G₂/M period in control group,20%ID₅₀of As₂O₃ group,radiation therapy group and radiation therapy plus 20%ID₅₀of As₂O₃ group was 12.45±1.88,18.91±1.65,18.42±2.29 and 31.07±6.66 respectively.The arrest effect was much more evident when As₂O₃ and iradiation were applied combinatively than either of them was used alone(P＜0.01,P=0.000).4.When As₂O₃ and iradiation was applied combinatively,the expression of p21^waf1/cip1protein was increased much more than that of either of them used alone. While the expression of Bcl-2 protein was decreased on the counterparts.Conlusion: 1.As₂O₃ had radiosensitizing effect on Hela cells of Human cervical Carcinoma.2.The mechanism of As₂O₃ enhance radiosensitivity on Hela cells may be inhibiting cells repairing,changing the cell cycle and up-regulating the expression of p21^waf1/cip1protein and down-regulating the expression of Bcl-2 protein.