Effects Of Dihydroartemisinin Combined With Irradiation On Human Laryngeal Squamous Carcinoma Cells

Objective: To study the effect of Dihydroartemisinin（DHA）on proriferation and radiosensitization when combined with irradiation,and cell cycle changes in laryngeal squamous cell carcinoma Hep-2 cells,in order to define the possible role of Stat3 in these processes.Methods:1 The inhibitory effects on Hep-2 cells of different concentrations of DHA（10μM,20μΜ,40μM,80μM,160μM,320μM）at 24 or 48 hours was detected by MTT assay.2 The radiosensitivity of Hep-2 cells treated with 20μM DHA and increasing radiation dose（0Gy,2Gy,4Gy,6Gy）was detected by clonogenic assay,and the sensitizing enhancement ratio（SER）of Hep-2 cells was measured with multi-target single hit module.3 Cell cycle changes of Hep-2 cells treated with 20μM DHA combined with 6Gy radiation were measured using flow cytometry.4 The protein expression of Stat3,p-Stat3 and Cyclin-D1 in Hep-2 cells treated with 20μM DHA combined with 6Gy radiation was analysed by Western blot analysis.Results:1 Cytotoxic effects of DHA on Hep-2 cells are evident.The inhibition ratio at different concentrations of DHA（10μM,20μM,40μM,80 μM,160μM,320μM）was 0.171±0.016,0.215±0.019,0.308±0.033,0.523±0.061,0.832±0.028,0.904±0.01 at 24 hours,and 0.197±0.010,0.272±0.029,0.382±0.034,0.742±0.030,0.870±0.013,0.934±0.005 at 48 hours,respectively.The inhibition rate was increased significantly with increased concentrations of DHA and treated time intervals（P<0.05）.IC₅₀ at 24h and48h was 58.48μM and 41.98μM,respectively.2 DHA increased the radiosensitivity of Hep-2 cells at 20μM.SER was2.091±0.232.SF was 0.474±0.023,0.109±0.012,0.026±0.009,respectively when treated by 2Gy,4Gy,6Gy irradiation only,and 0.243±0.020,0.035±0.002,0.006±0.002 when treated with DHA combined with 2Gy,4Gy,6Gy irradiation,they all decreased significantly contrasted with the irradiation-only group（all P < 0.05）.D₀,D_q and D₃₇ was 1.243±0.073,1.283±0.082 and 2.525±0.024,when treated by irradiation only,and1.013±0.050,0.638±0.115,1.651±0.065 when treated with DHA combined with irradiation,all decreased significantly contrasted with the irradiation-only group（all P<0.05）.3 DHA combined with irradiation had effect on the cell cycle of Hep-2cells.When treated with DHA only,contrasted with control,there was G₀/G₁ phase arrest（P<0.05）,G₂/M phase decrement（P<0.05）,and no significant change on S phase（P < 0.05）was noted.When treated with DHA plus irradiation,contrasted with irradiation only,there was G₀/G₁ phase arrest（P<0.05）,S phase arrest（P<0.05）,and G₂/M phase decrement（P<0.05）.4 Protein expression of Stat3 remained unchanged under any treatment（P< 0.05）.And expression of p-Stat3（Tyr705）,Cyclin-D1 decreased when treated with 20μM DHA with or without irradiation（P<0.05）.However,the expression of p-Stat3 increased after 6 Gy irradiation（P<0.05）.Conclusions:1 Cytotoxic effects of DHA on Hep-2 cells increased significantly with the increase of DHA concentration and treatment time.2 DHA promoted the radiosensitivity in Hep-2 cells.3 DHA induced G₀/G₁ phase arrest in Hep-2 cells with or without irradiation,and inhibited the G₂/M phase transition induced by radiation.4 By affecting Stat3 signal transduction pathway,DHA downregulated the phosphorylation level of Stat3 at Tyr705 site,resulting lower expression of Cyclin-D1,in which way DHA affected the cell cycle and apoptosis in Hep-2 cells.It was also found that,irradiation influenced protein expression levels of p-Stat3,which were increased after treatment with 6 Gy of irradiation,and suppressed by DHA to some extent.