| Part 1Effects of Suberoylanilide Hydroxamic Acid on the Radiotherapy Sensitivity,Cell Cycle and Apoptosis on the Pancreatic CancerObjective:To investigate the role of SAHA on the radiotherapy sensitivity,cell cycle and apoptosis of Capan-2 and Panc-1 cells.Methods:(1).The survival rate and IC50 of Capan-2 and Panc-1 cells were determined by CCK-8 after treated with 0.5,1,2,4,6,8,10,12 umol/L of SAHA with 24h,48h or 72h;(2).The cloning experiments assay was performed to determine the radiation-sensitizing effect of SAHA on Capan-2 and Panc-1 cells with the concentration of less than 20%IC50(0.5 umol/L)SAHA combined with radiation(0,2,4,6,8 Gy);(3).Analyzing the distribution of cell cycle by flow cytometry(FCM);(4).Analyzing the distribution of cell apoptosis by flow cytometry;Morphology changes of Capan-2 and Panc-1 cells were also observed after staining with Hoechst33528.In methods(3),(4):The cells was treated in vitro with no treatment,SAHA(0.5 umol/L)alone,radiation(2Gy)alone and SAHA combined with radiation.Results:Inhibition of cell proliferation seemed more dependent on the dose and the time.The IC50 values of SAHA for 48h in Capan-2 and Panc-1 cells were 3.012umol/L and 4.223umol/L,respectively.The value of D0,Dq in group of SAHA combined with radiation was obviously lower than of radiation alone group.The SER were 1.296 and 1.100,respectively.Cell cycle analysis showed that the cell population in G2/M phase was increased,the percent of apoptosis cells was apparently increased(p<0.05).There were higher percentages of bright blue cells(apoptosis)after treated with SAHA combined with radiation.Conclusion:SAHA could inhibit the proliferation,enhance the radiotherapy sensitivity,induce the apoptosis and make the cell cycle arrested at the G2/M phase in Capan-2 and Panc-1 cells.Inducing G2/M phase block and apoptosis may be one of the mechanism of radiotherapy sensitivity.Part 2Possible Mechanisms of Suberoylanilide Hydroxamic Acid’s Radiotherapy Sensitivity on the Pancreatic CancerObjective:To investigate the mechanisms of SAHA enhancing Panc-1 cell’s radiotherapy sensitivity,including the pathway of DNA double-stand breaks(DSBs)and apoptosis.We analyzed Rad51 gene related to radiosensitivity so as to make further research for molecular target and mechanism in radiosensitizing,provided the theoretical basis for clinical application of SAHA.Methods:(1).Detection the relation between DSBs repair gene and apoptosis related gene.Western Blot(WB)were used to detect the expression of Bax,Bcl-2,Ku70,Ku86,Rad51,Rad54;(2).RAD51-shRNA infected Panc-1 cells so as to silence RAD51 gene expression;(3).Real-time Quantitative PCR(qPCR)were used to detect the efficiency of RAD51 gene silencing;(4).The cloning experiments assay was performed to observe the concentration of less than 20%IC50(0.5 umol/L)SAHA combined with radiation(2Gy)on Panc-1 cells infected by RAD51-shRNA.In methods(1),(4):The cells was treated in vitro with no treatment,SAHA(0.5 umol/L)alone,radiation(2Gy)alone and SAHA combined with radiation.Results:SAHA combined with radiation induce Panc-1 cell apoptosis by down regulating the expression of bcl-1 and up regualating the expression of bax.SAHA combined with radiation can increase the radiosensitivity of Panc-1 cell by inhibiting the expression of Ku70,Ku86,Rad51,Rad54.RAD51-shRNA can stable The Panc-1 cell was infected by RAD51-shRNA steadily,and the experiment of qPCR showed that RAD51 gene had been silented efficiently.The concentration in group of SAHA combined with radiation was obviously lower than of radiation alone group(p<0.05)in Panc-1 cell.But for the Panc-1 cell which was short of RAD51 gene,the concentration in group of SAHA combined with radiation had no difference with radiation alone group(P>0.05).Conclusion:SAHA has influences on the pathway of DSBs repair and apoptpsis simultaneously,SAHA increase the radiosensitivity of Panc-1 cells with the interaction of the two factors. |
PDF Full Text Request