| ObjectivesIn the world,breast cancer is one of the most common malignant tumors in women,which is a serious threat to the health and life of women.According to WHO statistics,the incidence and mortality rate of breast cancer in China is lower than the global average level,but the incidence of breast cancer is increasing rapidly in recent years.At present,the main clinical methods for the treatment of breast cancer are surgical resection,chemical therapy and radiation therapy.How to effectively treat breast cancer has become the focus of the attention.Hydroxyurea(HU)is an ribonucleotide reductase inhibitor and has been employed for the treatment of melanoma,head and neck cancers in addition to other cancers.Valproic acid(VPA)is a kind of histone deacetylase inhibitors(HDACis),and it not only can inhibit the growth of breast and other cancer cells,but also can sensitize the tumor cells to ionizing radiation(IR)treatment.Multiple reports suggested that the combination of VPA and HU can trigger apoptosis to cooperatively kill tumor cells.However,the molecular mechanism of the combination of HDACis and ribonucleotide reductase inhibitor for anti-tumor effect remains unclear.Replication protein A(REPA)is a single-strand DNA(ssDNA)-binding protein,which is a heterotrimer containing three subunits,REPA1,REPA2 and REP A3.Recent reports have demonstrated that REPA plays an important role in regulating multiple cellular functions involving DNA metabolism,such as DNA replication,cell cycle checkpoint activation and DNA repair.In particular,REPA2 can be phosphorylated by phosphatidylinositol 3-OH-kinase-related kinase family comprising ataxia-talangiectasia mutated(ATM),ATR and DNA-dependent protein kinase in response to DNA damage or during cell cycle process.A few studies have indicated that REPA2-p is required for DNA DSB repair,regulation of DNA mitotic process and cell cycle checkpoint.REPA2-p can interact with recombinase Rad51 for the regulation of HR activity in vitro.Further studies clearly show that the REPA2-p-mediated HR pathway is specifically involved in the repair of HU-induced DNA replication arrest but not IR-induced DNA DSBs.Thus,our data demonstrated that VPA and HU synergistically suppressed tumor cells via disturbing REPA2-p-mediated DNA repair pathway,which provides a new way for combining chemotherapeutic drugs to sensitize breast cancer cells.Methods1.Establishment of a cell line working model for expressing homologous recombinant substrate pDR-GFPThe plasmid containing pDR-GFP was transferred into EUFA423 cells by Lipofectamine 2000 and Opti-MEM,and EUFA423 cells stably expressing the pDR-GFP plasmid(EUFA423/pDR-GFP)were selected by DMEM medium containing puromycin.The cells that can emit green fluorescent protein were detected by flow cytometry,and EUFA423 cells that can stably express the homologous recombination substrate pDR-GFP were further screened.2.Acquisition of primary culture-carcinoma cells of primary breast cancer in rats50 days old female SD rats were sensitized with dimethyl-benzanthracene(DMBA),and approximately 90 days later,the rats were induced to develop primary breast cancer.The rat primary breast cancer tissue was removed in a sterile environment.A portion of the breast cancer tissue was cut and used for primary cell culture.The other part of the breast cancer tissue was fixed with 10%formalin and HE staining was performed to identify the character of the tissure.3.Drug treatment of cancer cellsThe breast cancer cell line EUFA423 was used in this study.And the cells were divided into four treatment groups:blank control group,VPA alone group,HU alone group,VPA and HU combined group.In the VPA alone group,EUFA423 cells were treated with 0.5 mM VPA for 24h or 48h;HU alone group was used to treat EUFA423 cells with 2 mM HU for 18h;VPA and HU combined groups were first treated with 0.5 mM VPA for 24h or 48h,after which the EUFA423 cells were further co-treated with 0.5 mM VPA and 2 mM HU for 18h.4.The detection of DNA DSBsAfter each group of cells were treated with the drugs,EUFA423 cells with good growth status and logarithmic growth phase were used to prepare cell suspensions,then neutral and alkaline comet assays were performed.The Nikon ELIPSE Ti-U inverted fluorescence microscope was used to observe the tails of the cells in each group and photographed,and the tails of each group were quantitatively analyzed using the cometscore software.After treating cells of each group with the drugs,cell proteins of each group were collected and the expression of yH2AX protein and its foci were detected in each group by immunoblotting and immunofluorescence assay,respectively.The differences in DNA DSBs between cells in each group were reflected by the trailing distance of cells,the expression of yH2 AX protein and its foci.5.The effect of DNA DSBs on the survival of breast cancer cells by the test of cell cloning formationWell-growing EUFA423 cells in a logarithmic growth phase were seeded in 60 mm petri dishes and three replicates were set in each group.After treatment with the drug,incubation was continued for 14 days until macroscopic cell colonies appeared at the bottom of the culture dish.When each colony was>50 cells,the culture was terminated.Cell clones stained with crystal violet were counted and the colony formation rate of each group was calculated.6.The effect of drug treatment on the ability of DNA DSBs repair in breast cancer cellsAfter the termination of drug treatment in each group of cells,fresh culture medium was used to continue culture for 24h so that the cells could repair the DNA DSBs damage caused by the drug through their own repair ability.After that,cell proteins of each group were collected and the expression of ?H2AX protein and its foci were detected in each group by immunoblotting and immunofluorescence assay,and the ability of repairing DNA DSBs damage in each group within 24h was obtained.7.HR assayThe cells of EUFA423/pDR-GFP was treated by the drug first,Then the treated cells were collected and made into a cell suspension.The HR repair efficiency of each group of cells was detected by flow cytometry.8.Cell cycle analysisSince the HR repair function of cells is closely related to the cell cycle,especially the S/G2 phase,so cell cycle profiling was further examined in oreder to exclude whether the drug action has an effect on the cell cycle.Drug treatment of EUFA423 cells,30min before the termination time,each group of media were incorporation of 10?M BrdU,then collected cells were made into a suspension,fixed with 70%ethanol,and stored at-20 ? overnight.The incorporation of BrdU in each group of cells was detected by flow cytometry,and the cell cycle results of each group of cells were obtained.9.The detection of related proteins in HR repair pathwayAfter treatment of EUFA423 cells with the drugs,the proteins of each group were collected and subjected to immunoblot experiments to detect the expression of REPA2-p and Rad51 proteins associated with the HR repair pathway in each group.The relationship between REPA2-p protein and Rad51 protein was further explored by co-immunoprecipitation(Co-IP).After treatment of EUFA423 cells with the drugs,the cells were fixed with 4%formaldehyde,and the foci formation of REPA2-p and Rad51 in each group was observed and counted by immunofluorescence staining.Results1.VPA at safe concentrations can result in the accumulation of more DNA DSBs after HU treatment.Neutral comet assay and alkaline comet assay showed that compared with the control group,the cell tail of the VPA-treated group had a slight increase,and the tail distance of the HU-treated group increased significantly,and the VPA and HU combined effect cell tails further increased.The increase in VPA and HU combination group was the most significant(P<0.05).Western blotting results showed that there was no accumulation of DNA DSBs in EUFA423 cells without both VPA and HU treatment since the expression of yH2AX in the cells was not detected.After treatment with HU alone,the expression of yH2AX protein in EUFA423 cells was significantly increased.The expression of ?H2AX protein was not significantly increased in VPA 24h-pretreated group and HU group compared with HU alone group.However,when the pretreatment time of VPA was prolonged to 48h,the yH2AX protein expression in the combined group increased 1.4-fold(P<0.05)as compared with HU alone group.The results of immunofluorescence showed that only a small amount of EUFA423 cells showed yH2AX focus in the VPA alone group.The number of cells containing yH2AX focus in HU alone group increased by a factor of 3.0(P<0.05)compared with the control group alone.After 24hor 48h of VPA pretreatment and further combination treatment with HU,the number of cells containing yH2AX increased by 4.5-fold and 4.8-fold,respectively,as compared with the control group(P<0.05).2.VPA at safe doses can sensitize breast cancer cells to HU treatment.The results of colony formation assay showed that compared with the control group,cell survivsal was decreased by 19.55%and 35%(P<0.05)when VPA alone was used for 24h or 48h,and cell survival was decreased by 52.63%when HU was used alone(P<0.05).).After 24h or 48h of VPA pretreatment and further combination treatment with HU,the cell survival was decreased by 70.62%and 81.04%respectively(P<0.05).Because VPA also caused a significant decrease in cell survival,the elimination of VPA and HU in combination with a significant reduction in cell viability may be a superposition of the two drugs rather than the synergy.We standardized the results of the experiment.After the standardization,the combined effect of the two drugs still showed a significant decrease in cell viability compared to the HU alone(P<0.05),indicating that the combination of VPA and HU decreased the survival rate of EUFA423 cells as a synergistic effect.A safe dose of VPA can increase the sensitivity of EUFA423 cells to HU.3.Successful establishment of cell line working model for HR detection.The results of flow cytometry showed that EUFA423 cells expressing the homologous recombination substrate pDR-GFP were successfully selected through DMEM medium containing puromycin and the EUFA423/pDR-GFP cell line was established.4.VPA can disrupt HR activity in response to HU-induced replication arrest.The amount of yH2AX protein in the cells after 24h recovery was detected by Western blotting.The amount of ?H2AX protein in the VPA-treated group was not decreased,indicating that the intracellular DNA DSBs were almost repaired.The expression of yH2AX protein was also significantly reduced in the 24h HU group(P<0.05).However,the expression of ?H2AX protein in the combined groups of VP A and HU was still higher(P<0.05),indicating that the ability of DNA and DSBs in two drugs combined group repaired was significantly reduced.The percentage of cells containing yH2AX foci in each group was counted by immunofluorescence assay.After 24h of recovery,the percentage of cells containing yH2AX foci in HU-treated group was increased by 2.1 times compared with the control group(P<0.05),which was significantly lower than before the recovery.After 24h of recovery,compared with the control group,the percentage of cells containing ?H2AX foci in VPA and HU combination group was increased by 4.3-fold and 4.9-fold,respectively,compared with the control group(P<0.05),which was the similer as before the recovery.5.VPA at safe dose can disrupt HR activity in response to HU-induced replication arrest without the relationship of cell cycle.Flow cytometry was used to detect the efficiency of HR repair in each group of EUFA423/pDR-GFP cells.The efficiency of HR repair in the control group of EUFA423/pDR-GFP cells was 74.2*10-6;the HR repair efficiency of EUFA423/pDR-GFP cells in the VPA-treated group was reduced to 61.3*10-6(P<0.05);however,the HR repair efficiency of/pDR-GFP cells in HU-treated group alone was significantly increased to 141.9*10-6(P<0.05);and when VPA and HU were combined,the HR efficiency of EUFA423/pDR-GFP cells was significantly reduced to 100*10-6 as compared with the HU-treated group alone(P<0.05).The results of flow cytometry to detect the cell cycle reflected by BrdU incorporation showed that compared with the control group,the HU-treated group and the two drugs combined group significantly increased the proportion ofEUFA423 cells in S phase,but there was no significant difference between the two drugs combined group and individual HU group as compared with other group in the accumulation of EUFA423 cells in S phase.Combined with the previous experimental results,even though the combined effect of VPA and HU also showed accumulation of EUFA423 cells in S phase,the combined effect of VPA and HU can still significantly reduce the HR repair ability of EUFA243 cells compared with HU alone group.VPA inhibited the HR repair function of HU-induced replication fork block in EUFA423 cells regardless of cell cycle.6.VPA at safe dose inhibited the function of REPA2-p and Rad51 after HU-induced DNA replication arrest.The expression of REPA2-p protein in each group of cells was detected by Western blotting.We found that HU induced higher phosphorylation of REPA2 in EUFA423 cells.After VPA pretreatment,combined with HU,the highly phosphorylated REPA2 protein expression was significantly reduced(P<0.05).From immunofluorescence experiments,we get the same trend.The expression of Rad51 protein in each group was detected by Western blotting.The results showed that there was no significant difference in the expression of Rad51 protein in each group.Immunofluorescence experiments further showed that compared with the control group,the percentage of cells expressing Rad51 foci was slightly decreased in the VPA-only group(P<0.05);the percentage of cells expressing the Rad51 foci was significantly increased in the HU-treated group alone with 126.1%;compared with the HU-treated group,VPA and HU combined group,the percentage of cells expressing Rad51 foci was significantly reduced by 35.7%(P<0.05).Since the activity of Rad51 protein corresponds to the formation of foci in the nucleus,even though there is no significant difference in the expression of Rad51 protein in each group of cells,the percentage of cells containing Rad51 foci in the nucleus of each group of cells shows a significant difference,in another word,the activity of Rad51 protein in each group showed significant differences.Co-IP results showed that there are interactions between REPA2-p and Rad51 and that both proteins are present in the same protein complex precipitate.7.Effect of safe dose of VPA combined with HU on primary culture cells from DMBA-induced breast cancer tussure in rats.The tissue was confirmed as breast cancer of rat by pathological morphology.Cell clone formation experiments showed that the combined effect of VPA and HU also significantly reduced the survival rate of primary chiture-breast cancer cells in rats.The percentage of cells expressing yH2AX in each group was detected by immunofluorescence.The results showed that the VPA-alone and HU-treated groups only slightly increased the percentage of cells expressing the yH2AX foci,while the percentage of cells expressing the yH2AX focus in VPA and HU combined groups significantly increased(P<0.05).After treated cells in each group were recovered for 24h with fresh medium,the percentage of cells expressing yH2AX foci in VPA alone and HU alone group significantly decreased,while the percentage of cells expressing the ?H2AX foci in VPA and HU combined groups remained at a higher level.Conclusions1.A safe dose of VPA can cause further accumulation of DNA DSBs in the nuclei of breast cancer cells EUFA423 caused by HU,and increase the sensitivity of breast cancer cells to HU;2.A safe dose of VPA combined with HU inhibited the growth of breast cancer cells by inhibiting REPA2-Rad51-mediated HR repair pathway;3.The safe dose of VPA increased the sensitivity of breast cancer cells to HU regardless of cell cycle.4.In primary culture cells from the tissure of DMBA-induced breast cancer in rats,the combination of a safe dose of VPA and HU resulted in the accumulation of DNA DSBs in the nucleus,the ability of cells to repair DNA DSBs was weakened,and cell survival was reduced. |
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