The Molecular Mechanism Of The Radiosensitization Induced By Valproic Acid In Breast Cancer Cells Is P53-dependent

ObjectiveBreast cancer is one of the most common malignant tumors that seriously threaten women’s life safety and physical health.Ionizing radiation(IR)is a common method for clinical treatment of breast cancer patients,finding effective radiosensitizers has always been the focus of the field.IR can cause serious DNA damage,that also means the DNA double-strand breaks(DSBs)damage,homologous recombination(HR)plays an important role in DSBs repair mechanism.Valproic acid(VPA)is a short-chain fatty acid with an anti-tumor activity.Our and other research groups have found that 0.5 mM VPA can inhibit the growth of tumor cells and increase the radiosensitivity of breast tumor cells by interfering with BRCA1-RAD51-mediated HR repair mechanism.The wild-type tumor suppressor gene p53(wtp53)has an inhibitory effect on HR repair to maintain HR function at a nonnal level Studies have shown that in colorectal cancer cells,p53 affected the radiosensitization of VPA by interfering apoptosis mechanism,which showed that the radiosensitization of VPA was particularly significant in wtp53 expressing cells,while inhibited in defective p53(dp53)expressing cells.This study aims to investigate whether the radiosensitization of VPA is affected by p53-deficient cells in breast cancer cells and the relationship between the mechanism and HR repair function,to provide reliable theoretical guidance and experimental basis for the application of VPA for clinical treatment of breast cancer patients.Methods1.Establishment of p53 expression down-regulated isogenic pairing cell line working model and detection of p53 expressionInfection of breast tumor cells MCF7 with lentiviral particles solution expressing PLKO.1 and p53 shRNA to establish anisogenic pairing cell line expressing wtp53 and dp53;MCF7 cell was used as a positive control and detected the expression of p53 by western blotting to identify the establishment situation of p53 down-regulatedisogenic pairing cell line(wtp53and dp53).Meanwhile,wtp53 and dp53 cells were divided into four groups respectively:control group,VPA group(0.5 mM VPA treatment for 24 hours alone),IR group(8GyIR alone),and VPA+IR group(VPA pretreatment for 24 hours combined with 8Gy IR).Western blotting was used to detect the expression of p53 protein in wtp53 and dp53 cells with different treatments again.2.Detection of DNA DSBs in nuclearThe wtp53 and dp53 cells atlogarithmic growth phase were divided into four groups respectively:control group,VPA group(0.5 mM VPA treatment for 24 hours alone),IR group(8Gy IR alone),VPA+IR(0.5 mM VPA pretreatment for 24 hours combined with 8Gy IR).Two parts were prepared:one part of cells recovered for 30 minafter IR treatment,and the cells of each group were collected to prepare single cell suspension for neutral comet assay,the other part recovered for 6 hours after IR treatment to prepare a single cell suspension for immunofluorescence experiments.The nuclear tail length and nuclearγH2AX focus formation in each group was detected respectively to analyze the DNA DSBs damage in the nucleus.3.Detection of cell proliferation viabilityThe wtp53 and dp53 cells atlogarithmic growth phase were divided into eight groups respectively:control group,VPA group(0.5 mM VPA alone for 24 h),IR group(three groups:2,4,6Gy alone),VPA+IR group(three groups:0.5 mM VPA pretreatment 24 h after combined with 2,4,6 Gy IR).After treatment,the fresh medium was replaced and cells were cultured for 14 days to terminate.After the crystal violet staining,the percentage of colony formation of each group of cells was calculated to reflect the proliferation and survival ability of the cells.4.Detection of HR efficiencyThe HR substrate reporter gene pDR-GFP carrying the green-labeled fluorescent protein was transferred into the breast tumor cell MCF7,MCF7 cells stably expressing pDR-GFP were selected.MCF7/pDR-GFP cells were infected with lentiviral particles solution expressing PLKO.1 or p53 shRNA to establish MCF7/pDR-GFP/wtp53 andMCF7/pDR-GFP/dp53 cells,after transfected with endonuclease I-sceI for 4 hours,cells were planted in p60 according to the total number of 2-3*105.The two cells were divided into two groups respectively:control group,VPA group(0.5 mM VPA treatment for 24 hours).After treatment,fresh cell culture medium was replaced.After 72 hours,cells were digested by trypsin and centrifugated at 1000 r/min for 5 min,then discard the supernatant,after resuspend by PBS,105 cells were detected by flow cytometry and counted the number of cells emitting green fluorescence,homologous recombination(HR)repair efficiency =number of green fluorescent cells/(1*105),compared the HR efficiency of each group.5.Detection of HR repair proteins BRCA1 and RAD51The wtp53 and dp53 cells at logarithmic growth phase were divided into control group,VPA group(0.5 mM VPA treatment for 24 hours alone),IR group(8 Gy IR alone),and VPA+IR group(VPA pretreatment for 24 hours combined with 8 Gy IR).Cells were fixed after treatment,and the focus formation of BRCA1 and RAD51 in the nucleus were detected by irmmunofluorescence,and count the percentage of the cells with positive focus formation to the total number of cells.Results1.Establishment of p53 expression down-regulated isogenic pairing cell line(wtp53 and dp53)modelsAccording to the results of western blotting,compared with MCF7,the expression of p53 protein in MCF7 cells transfected with lentiviral particle PLKO.1 did not change,the expression of p53 protein significantly decreased in cells transfected with p53shRNA,and without p53 expression was observed after VPA or IR treatment,it was indicated that anisogenic pairing cell line with down-regulated p53 expression(wtp53 and dp53)was successfully established.2.p53 expression deficiency inhibits the promotion of VPA to IR-induced DNADSBs damage accumulation in cellsAccording to the results of comet assay,in cells expressing wtp53,compared with IR group,the tail length of nucleus in VPA+IR group increased by 1.82 times(P<0.05),it was indicated that VPA can further increase the DNA damage of cells induced by IR.In cells expressing dp53,the tail length in VPA+IR group was 1.23 times higher than that of the IR group,still significantly lower than that of the VPA+IR group cells expressing wtp53(P<0.05),it was indicated that deficiency of p53 protein expression might attenuate the accumulation of VPA to IR-induced DSBs damage in cells.According to the results of immunofluorescence experiments,the percentage of yH2AX-positive cells in VPA+IR group was significantly increased by 30.5%compared with IR group in cells expressing wtp53(P<0.05);In cells expressing dp53,compared with its IR group,the percentage of positive cells containing yH2AX focus formation in VPA+IR group was slightly increased,while still significantly lower than that of the VPA+IR group cells expressing wtp53 30%(P<0.05),it was also suggested that after the inhibition of p53 expression in MCF7 cells,the accumulation of VPA to IR-induced DNA DSBs damage in breast cancer cells was inhibited.3.Effect of VPA combined with IR on proliferation and survival of breast cancer cells with different expressions of p53According to the results of cell colony formation experiments,without IR treatment,in the cells expressing wtp53,the percentage of colony formation in the VPA group was 23%lower than the control group(P<0.05).In the cells expressing dp53,the colony formation rate of the dp53 cell control group was 50.3%higher than the control group cells expressing wtp53(P<0.05),VPA treatment was further given based on this,the percentage of colony formation in VPA group cells expressing dp53 cells was 13.6%lower than the control group(P<0.05),however,it was significantly 59.7%higher than VPA group cells expressing wtp53(P<0.05).After IR treatment,the corresponding groups without IR treatment were corrected as 100%,the cell survival of each group decreased significantly with the increase of IR.In the cells expressing wtp53,the colony formation rates of VPA+IR(2 Gy)groups were 8.4%lower than IR(2 Gy)group(P<0.05),the colony formation rates of VPA+IR(4 Gy)20.4%lower than IR(4 Gy)group(P<0.05).After treatment of IR,the colony formation rate of IR(2,4,6 Gy)groups were significantly higher than the IR group cells expressing wtp53(P<0.05).On this basis,VPA and IR were combined to treat cells expressing dp53,compared with the IR group,though the cell survival in VPA+IR group slightly decreased,however,it was still higher than that of VPA+IR group cells expressing wtp53(P<0.05).These results suggested that VPA could increase the sensitivity of cells expressing wtp53 to IR,however,the deficiency of p53 expression could attenuate the radiosensitization induced by VPA.4.The effect of VPA on HR repair ability of breast cancer cells depends on p53According to the results of flow cytometry,the control group of MCF7/pDR-GFP/wtp53 cells was corrected as 100%,compared with control group,the HR efficiency decreased by 51.6%after VPA treatment(,P<0.05),indicating that VPA inhibited HR efficiency in cells expressing wtp53.In MCF7/pDR-GFP/wtp53 cells,compared with the control group of MCF7/pDR-GFP/wtp53 cells,the HR efficiency in control group of MCF7/pDR-GFP/dp53 cells increased by 49%(P<0.05),suggested that p53 deficiency can increase HR repair efficiency;further VPA treatment was given based on this,HR efficiency decreased by 20.3%,but still 80.3%higher than VPA group of MCF7/pDR-GFP/wtp53 cells(P<0.05).In MCF7/pDR-GFP/dp53 cells,HR repair ability was over-enhanced to result in inhibition of radiosensitization of VPA on breast cancer.5.p53 effects the radiosensitization effect of VPA on breast cancer cells through the BRCA1-RAD51-mediated HR repair mechanismAccording to the results of immunofluorescence experiments,the protein focus formations of HR repair key proteins BRCA1 and RAD51 in the nucleus were determined.In the cells expressing wtp53,the percentage of BRCA1 and RAD51 focus formation positive cells in VPA+IR group decreased by 15.6%and 20.4%compared with IR group(P<0.05),suggested that IR-activated HR repair pathway was inhibited by VPA.In cells expressing dp53,the percentage of BRCA1 and RAD51 positive cells in IR group increased by 31.7%and 27.6%compared with IR group of wtp53 cells(P<0.05),HR repair wasover-enhanced;the percentage of BRCA1 and RAD51 focus formation positive cells in VPA+IR group were reduced slightly compared with IR group by combination of VPA and IR,while still significantly higher than 41.5%and 39.4%in VPA+IR group of wtp53 cells(P<0.05),HR function was still over-enhanced.The results indicated that deficiency of p53 inhibited the radiosensitization effect of VPA by over-enhancement of BRCA1-RAD51-mediated HR mechanism.Conclusions1.Deficiency of p53 expression inhibits more accumulation of IR-induced DNA DSBs in the nucleus of breast cancer cells induced by VPA.2.With the deficiency of p53 expression,the radiosensitization of VPA to breast cancer cells is inhibited.3.The mechanism of the decrease of VPA-dependent radiosensitization caused by p53 deficiency is related to the enhancement of BRCA1-RAD51-mediated HR repair function.