The Study On VPA-induced Radiosensitivity To Osteosarcoma Cells And Its Mechanism

ObjectivesOsteosarcoma is one of the malignant bone tumors in children and adolescents under 20 years old.In order to enhance radiation effect of tumor cells,finding an ideal radiation sensitizer is a hot spot in the field.Valproic acid(VPA)is the most representative drug of histone deacetylase inhibitors(HDACis),and it is widely used in clinical treatment of epilepsy and other spastic disease.Our and other research have shown that VPA could enhance the radio-sensitivity of some tumor cells,such as breast cancer cells,colon cancer cells and lung cancer cells.Our previous paper also revealed that the radio-sensitivity in breast cancer cells were associated with the dysfunction of DNA damage repair mechanism under the concentration of 0.3-0.8 mM of VPA,and the dysfunction of homologous recombination(HR)was the main mechanism.However,what the effect of HDACi may have on osteosarcoma cells is still unknown.Therefore,this study will explore the effect of VPA under the safe dose(0.5 mM)and safety critical dose(1.0 mM)on U20S cells,and investagate whether the effect of radio-sensitivity is related to HR and non-homologous end joining(NHEJ),and the purpose of this study would provide an important theoretical and experimental basis for radiation therapy of osteosarcoma clinical.Methods1.The detection of VPA-induced radiosensitivity in osteosarcoma cells by clonogenic survival assayFor the group of VPA alone,the cells were treated with 0.5 mM VPA for 24 h;U20S cell line was treated with 2,4 and 6 Gy of ionizing radiation(IR)respectively for the group of IR alone;U20S cell line in combination group was pretreated with 0.5 mM VPA for 24 h,and then irradiated with 0,2,4 and 6 Gy.The number of cell colonies(≥50 cells per clone)in each group was counted and cell survival was presented by the survival fraction(SF):SF =(the number of clones/seeded cells)/plating efficiency(PE).2.The analysis of VPA-induced DNA DSBs in U20S cells by neural comet assayFor the group of VPA alone,the cells were treated with 0.5 mM VPA for 24 h,U20S cell line was treated with 4 Gy of IR for the group of IR alone.U20S cell line in combination group was pretreated with 0.5 mM VPA for 24h,and then irradiated with 4Gy.The cells in each group were collected and made single cell suspension for the detection the length of DNA tail using the Trevigen Comet Assay kit.3.The analysis of the protein foci formation in response to DNA damage for VPA-induced DNA DSBs marker γH2AX and 53BP1,and HR-associated key protein BRCA1After the cells were irradiated for 6 h or 24 h,immunofluorescence assay was used to detect the foci formation of γH2AX and 53BP1,and BRCA1 in each group and the rate of protein foci formation in each group was calculated.4.The analysis of NHEJ repair frequencies and the NHEJ-associated main protein levelThe nuclease I-Scel plasmid was transfected into U20S cells containing expression of NHEJ recombination substrate to induce DNA DSBs,meanwhile the cells were treated with 0.5 mM or 1.0 mM VPA for 24 h,and then the cells were collected to detect the NHEJ repair frequencies with flow cytometry.Also,the cells in each group were collected for detecting protein expression of KU70,KU80 and DNA-PKcs by immunoblotting assay.5.The detection of genomic instability by Quantitative fluorescence in situ hybridization(Q-FISH)The treated cells in each group were incubated for 20 hours before 0.05 g/ml colcemid was added for a further 4 h incubation to obtain mitotic cells.1000 chromosome in each group were calculated to detect chromatid,chromosomal and radial structure aberrations.6.The detection of cell cycle profiling by flow cytometryU20S cells were treated with 0.5 mM or 1.0 mM VPA for 24 h.and cell cycle profiling were analyzed via flow cytometry.Results1.VPA can induce more DNA DSBs in osteosarcoma cells in response to IRWithout IR treatment,VPA alone showed a slightly longer DNA tail as compared with control group;IR alone and the combination with VPA and IR could obviously enhance the olive moments,and the olive moments in the combination group was the longest(P<0.05),At 30-min and 120-min post-IR,it was further found that the relative olive moments in combination group was obviously higher than IR alone groups by 14.02%(P<0.05)and 16.49%(P<0.05).The results from immunofluorescence staining assay showed that VPA at the concentration of 0.5 mM or 1.0 mM could induce yH2AX foci formation,the relative ratio increased 1.2-or 1.3-fold than the control group(P<0.05).After U20S cells were pretreated with 0.5 mM or 1.0 mM VPA for 24 h and then given 8 Gy radiation,at 6 h post-IR,the relative percentage of cells with yH2AX foci was increased to 1.6-or 2.1-fold that IR alone group(P<0.05).The relative ratio about 53BP1 foci in the cells pretreated with 0.5 mM or 1.0 mM VPA alone had an increase of 1.9-or 2.7-fold(P<0.05)as compared with control group,respectively.At 6 h post-IR,the relative ratio in the cells containing 53BP1 foci in combination of VPA and IR was obviously increased to 2.1-fold or 3.2-fold that IR alone group(P<0.05).2.VPA at safe dose can enhance the radiosensitivity of U20S cellsThe survival rate in the cells treated by VPA alone was decreased as compared with control group.There was a significant decrease of survival fraction in all of the combination with VPA and IR groups(2,4 and 6 Gy)as compared with corresponding IR alone groups(P<0.05),the difference in the groups was 23.3%,32.4%,43.6%,respectively.3.VPA at safe dose can inhibit HR repair activity in U20S cellsAt 24 h post-IR,the percentage of cells containing yH2AX foci in the combination treatment of 0.5 mM or 1.0 mM VPA with 8 Gy was significantly higher than the cells treated with 8 Gy alone(P<0.05).their difference was 1.27-or 1.62 fold,respectively.At 24 h post-IR.the positive percentage of-the cells containing IR-induced 53BP1 foci in the combination group of 0.5 mM or 1.0 mM VPA with IR was also obviously higher than IR alone group(P<0.05),the difference between the groups was 2.24-or 3.43-fold.At 6 h post-IR,the positive percentage of the cells containing IR-induced BRCA1 foci in the combination group of VPA with IR was obviously lower than IR alone group by 26.5%(P<0.05).And at 24 h post-IR,the percentage of the cells containing IR-induced BRCA1 foci in the combination group of VPA with IR was lower than IR alone group by 15.3%(P>0.05).The cell survival rates in the combined groups(0.5 mM VPA or 1.0 mM with 10μM ABT888)was significantly lower as compared with other groups(P<0.05).4.VPA at safe dose can inhibit NHEJ repair activity in U20S cellsThe cells treated with 0.5 mM or 1.0 mM VPA for 24 h showed a significant reduction of NHEJ frequencies to 63.8%(P<0.O1)or 61%(P<0.01)in relative to control group.There was no difference between 0.5 mM and 1.0 mM VPA groups(P>0.05).The results from the immunoblotting assay demonstrated that the protein expression of KU80 in the cells treated with 0.5 mM VPA for 24 h with or without IR was significantly decreased;however,DNA-PKcs and KU70 protein level had no significant change in all groups.5.VPA can not change the cell cycle profiling in U20S cellsThe cell cycle profiling in U20S cells treated with 0.5 mM or 1.0 mM VPA was not significant changed as compared with control group.6.VPA can increase genomic instability in U20S cellsThere was no difference of the number of the chromatid breaks(P>0.05)or the chromosome breaks(P>0.05)between control and IR alone groups,whereas IR can increase the number of the radical structure to 7.34 from 1.59 in the group without IR treatment(P<0.05).After pretreatment with 0.5 mM VPA and followed by 2 Gy IR,the number of the chromatid breaks were further increased to 17.24 from 4.57 in the VPA alone group(P<0.01);the number of the chromosome breaks showed a more significant increase with the percentage to 43.1%from 18.27%in VPA alone group(P<0.01);and the number of the radical structure showed the same tendency(P<0.01).Conclusions1.VPA at safe dose and safety critical dose can lead to more DNA DSBs accumulation in response to DNA damage.2.VPA at safe dose can inhibit the growth of U20S cells and increase the cell radiosensitivity.3.VPA at safe dose can increase the radiosensitivity by disrupting both BRCA1-mediated HR and KU80-mediated NHEJ repair pathways.4.VPA at safe dose can increase genomic instability in U20S cells.