| OBJECTIVE : To investigate the radiotherapy sensitization role of Cepharanthine(CEP)on Nasopharyngeal carcinoma cells(NPC)and its possible mechanism.For Cepharanthine can applied to clinical as Nasopharyngeal carcinoma radiotherapy sensitization agent provide theoretical basis.METHODS:(1)The human Nasopharyngeal carcinoma cell lines CNE-1 and CNE-2 were used.Cell counting kit-8(CCK-8)was used to detection Cepharanthine on the influence of Nasopharyngeal carcinoma cells proliferation.(2)Clone forming assay was carried out to observe the radiotherapy sensitization role of Cepharanthine on Nasopharyngeal carcinoma cells.And Colonies were counted with the naked eye,with a cut-off of 50 viable cells.The surviving fraction(SF)was calculated as mean colonies/(cells inoculated×plating efficiency).(3)Cell cycle and the rate of apoptosis of the human Nasopharyngeal carcinoma cells were determined by FACSauto flow cytometry,which are through Cepharanthine,radiation single acting,and Cepharanthine combined with radiation.And the results were analyzed by the multicycle software.(4)Western-blot technique detected the expression of RAD51 and XRCC1 protein after the Nasopharyngeal carcinoma cells were treated.RESULTS :(1)Cepharanthine could inhibit the proliferation of Nasopharyngeal carcinoma cells in a time and dose dependent manner.(2)The radiosensitivity parameters D0,Dq and SF2 were significantly reduced in irradiated cells pretreated with CEP.After combination treatment,mean lethal dose of radiation(D0,Gy)value decreased(from 2.7 to 2.22 in CNE-1 cells,and from 3.34 to 3.15 in CNE-2 cells),indicating an enhancement of radiation sensitivity of CEP on both cells.The value of the quasithreshold dose(Dq,Gy)also decreased(from 1.8 to 1.3 in CNE-1 cells,and from 2.96 to 1.94 in CNE-2 cells),suggesting CEP treatment led to the inhibition of cellular capacity to repair potentially lethal radiation damage.The sensitivity-enhancement ratio(SER)of CNE-1 and CNE-2 cells were 1.26 and 1.23,which shows that CEP has a significant radiosensitizing effect on both cells.(3)The number of cells in G2/M phase significantly increased in the 4 Gy radiation group and the 4 Gy plus CEP group.Apparently,the number in the combination group was more than those in the control group 、CEP group and the radiation group(score: 36.29±6.24 vs 9.64±4.98,14.35±2.8,19.19±1.93 in CNE-1 cells,p<0.05;36.43 ±12.59 vs 8.21 ±3.3,10.74±5.16,21.78±3.23 in CNE-2 cells,p<0.05).(4)apoptosis was significantly observed in the CNE-1 and CNE-2 cells when CEP were combined with 4 Gy than those in the control group and the radiation group(15.57±2.59 vs 4.4±1.81,10.97±0.45 in CNE-1 cells,p<0.05;13.23±2.7 vs 1.55±0.83,9.07±2.2 in CNE-2 cells,p<0.05).(5)We examined whether CEP can regulate the expression of DNA repair proteins(RAD51 and XRCC1)in CNE-1 and CNE-2 cell treated with CEP and radiation.The expression of DNA repair proteins was examined by western blot analysis.The results showed that Compared to IR alone and CEP alone,combination of CEP and IR dramatically caused a persistent decrease of RAD51 and XRCC1 in both cell lines(RAD51 protein ratio of grey value in CNE-1 cells:1.08±0.01 vs 1.3±0.07,1.42±0.03;XRCC1 protein ratio of grey value in CNE-1 cells: 0.50±0.07 vs 0.59±0.04,0.88±0.09.RAD51 protein ratio of grey value in CNE-2 cells: 0.54±0.05 vs 0.66±0.05,0.80±0.09;XRCC1 protein ratio of grey value in CNE-2 cells:0.52±0.01 vs 0.84±0.02,0.96±0.03.Comparison between groups P<0.05).CONCLUTION: CEP enhances tumor radioresponse through multiple mechanisms that may involve the halted cell cycle progression at G2/M phase and the inhibition of DNA repair after exposure to radiation. |
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