The Combined Effect Of PARP-1 Inhibitor And Ionizing Radiation On DNA Damage And Apoptosis Of BRCA Mutant Cells

Background and object:Breast cancer is one of frequent female malignant tumors in our country,and the incidence and mortality is trending to ascend.Radiotherapy has been considered to be an important part of the comprehensive treatment of breast cancer,which improves the curative effect and prognosis of breast cancer.But the breast cancer susceptibility gene(BRCA)in breath cancer cells has a certain radiation resistance to the ionizing radiation,whichis an important factor to restrict the efficacy and prognosis of breath cancer.In this paper,by discussing the effect of different irradiation doses on DNA damage and apoptosis of BRCA non-mutant cells(MDA-MB-231)and BRCA mutant cells(MDA-MB-436),as well as the role and regulation mechanism of poly ADP-ribose polymerase-1(PARP-1)and BRCA on repairing irradiation-induced damage of breath cancer DNA,in order to provide the experimental basis for improving breast cancer radiosensitivity.Methods:1.The MDA-MB-231 and MDA-MB-436 were irradiated respectively according to the irradiation doses:0Gy,2Gy,4Gy,6Gy,8Gy and 10Gy.After different irradiation doses,the apoptosis rate of MDA-MB-231 and MDA-MB-436 was tested by flow cytometry,and the number of foci formation was detected by ?-H2AX immunofluorescence focus assay,so as to detect the DNA damage.2.The MDA-MB-231 and MDA-MB-436 were grouped according to the control group(CTRL),ionizing radiation alone(IR),3AB alone(3AB),ionizing radiation combined with 3AB(IR+3AB),and the radiation dose was 8Gy,the drug concentration of 3AB was 10mm.After treatments of different factors,the radiosensitivity of MDA-MB-231 and MDA-MB-436 was evaluated by formatting colony assay,the number of foci formation was detected by ?-H2AX immunofluorescence focus assay in order to detect the DNA damage,and the apoptosis rate was tested by flow cytometry.Results:1.(1)Flow cytometry suggested that the apoptosis of breath cancer cells would increase with increasing of irradiation dose,and it would reach the highest in irradiation dose 8Gy(MDA-MB-231 19.220%,MDA-MB-436 21.245%).In the irradiation dose 10Gy,the apoptosis rate of both MDA-MB-231 cells and MDA-MB-436 cells was slightly lower than that in 8Gy(MDA-MB-231 17.285%,MDA-MB-436 18.975%),but it had no statistically significant.Compared with MDA-MB-436 cells,the apoptotic rate of MDA-MB-231 cells was much lower,and had statistically significant(P<0.05).(2)?-H2AX immunofluorescence focus assay showed that DNA double strand damage(DSB)became more and more serious whit the irradiation dose increased.The number of foci formation would reach the highest in irradiation dose 8Gy(MDA-MB-231 19.220%,MDA-MB-436 21.245%),but it had no statistically significant compared to irradiation dose 10Gy.Moreover,the DNA strand damage in MDA-MB-436 cells was more severe(P<0.05).2.(1)The formatting colony assay suggested that the radiosensitiviry of MDA-MB-436 more significantly increased than MDA-MB-231,and the PARP-1 inhibitor 3AB could further enhance the radiosensitiviry.(2)In the condition of the same treatment factors,?-H2AX immunofluorescence focus assay showed that the DNA double-stranded damage of MDA-MB-436 was significantly more than that of MDA-MB-231,3AB could further enhance the effect,and the DNA damage of MDA-MB-436 cells in the IR+3AB group was the most Obvious.(3)Flow cytometry showed that the cells in the IR+3AB group had the highest rate of apoptosis,the difference was statistically significant(P<0.05).At the same processing factors,compared with MDA-MB-231,the apoptosis rate of MDA-MB-436 was obviously increased(P<0.05).Conclusions:1.With the increasing of irradiation dose,DNA double strand damage and apoptosis rate became more and more serious,but it would have a plateau when it reached a certain dose.And the DNA double strand damage and apoptosis rate of BRCA mutant cells was more obviously increased.2.The DNA damage,apoptosisrate and radiosensetivity of the IR+3AB group were more obviously increased than the 3AB group and IR group.In the combined effect of PARP-1 inhibitor and ionizing radiation,the DNA damage,apoptosisrate and radiosensetivity of BRCA mutant cells were more significantly increased than that of BRCA non-mutant cells,this result suggested that PARP-1 inhibitor combined with ionizing radiation increased the DNA damage and apoptosis of BRCA mutant cells.