Radiosensitization Effect And Mechanism Of Rapamycin Combined With SAHA On Human Lung Adenocarcinoma A549 Cell

Objective: To observe the radiosensitization effects of Rapamycin combined with histone deacetyltransferase inhibitor suberoylanilide hydroxamic acid(SAHA)on human adenocarcinoma A549 cells and investigate the possible molecular mechanismMethods: 1.To determine the dose of Rapamycin combined with SAHA:Culture of A549 cells in vitro,different concentrations of rapamycin(0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L,1000 nmol/L)or different concentrations of SAHA(0?mol/L,2.5?mol/L,5?mol/L,10 ?mol/L)or cross-over concentrations of rapamycin and SAHA were combined for culturing A549 cells for 24 hours,cell proliferation were measured by MTT;the ten percent inhibitory concentration(IC10)of two drugs on A549 cells for 24 hours was calculated.2.To observe the radiosensitization effects of Rapamycin combined with SAHA on A549 cells:A549 cells were treated with IC10 for 24 h,then given X-ray irradiation of 4 Gy.The survival fraction(SF)value was detected by cell colony formation assay.3.Study on radiosensitization effect of Rapamycin combined with SAHA(1)To detect the effects of Rapamycin combined with SAHA on the process of DSBs repair of A549 cells after radiation:A549 cells were treated with IC10 for 24 h,then given X-ray irradiation of 4 Gy.?-H2 AX protein and ?-H2 AX foci were detected by Western Blot and immunofluorescence staining after radiation 1h and 24h;Rad51,Ku80,Ku70 protein were detected by Western Blot after irradiation 4h;Rad51 m RNA?Ku80 m RNA?Ku70 m RNA were detected by RT-q PCR after radiation 4h.(2)To detect the effects of Rapamycin combined with SAHA on autophagy and acetylation of A549 cells after radiation:A549 cells were treated with Rapamycin or/and SAHA for 24 h,and X-ray irradiation of 4 Gy.The formation of autophagosome of A549 cells was observed by transmission electron microscope;LC3,p62 were detected by Western Blot.A549 cells were treated with SAHA or Rapamycin+SAHA for 24 h,Acetyl-Histone H3 protein(Ac-H3)were detected by Western Blot.Results: 1.To determine the dose of Rapamycin combined with SAHA:Both Rapamycin and SAHA possessed the suppression of the growth of A549 cells in a dose dependent manner;compared with the single drug group,cell proliferation of A549 cells was markedly inhibited by combination of Rapamycin and SAHA(p < 0.05).The IC10=Rapamycin:100 nmol/L;SAHA:2.5 ?mol/L.2.Radiosensitization effects of A549 cell were influenced by Rapamycin combined with SAHA:Colony formation assay was detected that the survival fraction(SF)in CTL group,IR group,IR+R group,IR+S group,IR+R+S group were 100.0±0.00%,67.8±6.32%,43.1±7.18%,58.5±1.17%,21.1±7.01%,compared with IR group,the degree of decrease in IR+R+S group was the most significant(p<0.05).3.The process of DSBs repair of A549 were influenced by Rapamycin combined with SAHA:(1)Compared with the CTL group,Western Blot and immunofluorescence staining were detected that the expression of ?-H2 AX protein and ?-H2 AX foci in each treatment groups were increased after radiation 1h(p<0.05),there was no significant difference between each group(p>0.05).The expression of ?-H2 AX protein and ?-H2 AX foci were decreased in each groups after radiation 24h(p<0.05),the decline in IR+R+S group was the narrowest.(2)Compared with the CTL group,Western Blot was detected that the expression of Rad51 protein was increased(p<0.05),the expression of Ku80 and Ku70 protein was no significant difference(p>0.05).Compared with the IR group,the expression of Rad51 protein in IR+R group and IR+R+S group was decreased(p<0.05),the expression of Ku80 protein in IR+R group and IR+S group and IR+R+S group was decreased(p<0.05),the expression of Ku70 protein in IR+R+S group was decreased(p<0.05).Compared with each group,the expression of Rad51,Ku80,Ku70 protein decreased in IR+R+S group was the most significant(p<0.05).(3)Compared with the CTL group,RT-q PCR was detected that the expression of Rad51,Ku80,Ku70 m RNA in IR groups and IR+R group were increased after radiation 4h(p<0.05),there was no significant difference between the two group(p>0.05).Compared with IR groups,the expression of Rad51,Ku80,Ku70 m RNA in IR+S group and IR+R+S group were increased after radiation 4h(p<0.05),there was no significant difference between the two group(p>0.05).4.Autophagy and acetylation of A549 cells were influenced by Rapamycin combined with SAHA:(1)Electon microscopy detected that Rapamycin and radiation can increase autophagosome formation of A549 cell,the effect of Rapamycin was the most remarkable.Western blot detected that Rapamycin and radiation can induce the expression of LC3 II protein increased,the ratio of LC3II/I was increased,the expression of p62 decreased.(2)Western blot detected that SAHA can induce the expression of Ac-H3 protein increased.Conclusion: 1.Rapamycin combined with SAHA can increase radiation sensitivity of A549 cell.2.The mechanism of Rapamycin combined with SAHA increased radiation sensitivity of A549 cell possibly inhibited the process of DSBs repair by up regulation of autophagy degrade the acetylated DSBs repair protein.