| ObjectivesBreast cancer has become the primary problem that threatens women's health.Chemotherapy has been the main method for treating breast cancer patients.In order to enhance the killing effect of chemotherapeutic drugs on tumor cells,drug combination is a commonly used clinical antitumor treatment strategy.The ideal drug combination means that on the premise of improving the ability to kill tumor cells,it can also reduce the toxic effect of the drug on normal tissues.However,such an ideal combination is still rare.The experimental results of us and other research groups have proved that a representative drug among histone deacetylase inhibitors(HDACi),Valproic Acid(VPA),is a drug for epilepsy At a safe dosage(300-800?M)in blood,it can not only inhibit the growth of a variety of breast tumor cells,but also enhance the ribonucleotide reductase inhibitors(RRi)chemotherapeutic drugs Hydroxyurea(HU)to kill leukemia,head and neck tumor cells and breast tumor cells.In view of the large doses,strong toxic and side effects required for VPA to work,there is an urgent need to develop highly effective and low toxicity VPA derivatives.2-hexyl-4-pentynoic acid(HPTA)is a kind of VPA derivatives that can also inhibit histone deacetylase(HD AC)activity,which affects HD AC activity.The IC50 dose is significantly lower than VPA,and there is no obvious liver and kidney damage,and the function is stronger than VPA.However,it has not been reported whether HPTA also has stronger antitumor effect than VPA and sensitizes HU.In addition,previous studies have demonstrated that in the DNA replication fork block damage caused by HU,the molecular mechanism of homologous recombination(HR)mediated by highly phosphorylated DNA replication protein A2-hyperphosphorylation(RPA2p)(RPA2p-HR)can specifically participate in the repair process of this damage.And in this process,RPA2p and Rad51 protein work together to regulate the homologous recombination activity of cells in vitro.Therefore,this project explored whether HPTA,a derivative of VPA,also has the ability to increase the killing effect of HU on breast cancer cells,and whether its molecular mechanism is related to the RPA2p-HR repair pathway.The purpose is to find more effective and low toxicity chemotherapeutic sensitizers,and to provide reliable experimental evidence and theoretical guidance for the combined application of HDACi and RRi drugs in the clinical accurate treatment of breast tumors.Methods1.The effect of VPA and HPTA on HU's inhibition of tumor cell proliferation at the same dose was detected by MTT test.The cells in the logarithmic growth phase were selected and seeded in 96-well plates,and the groups were set up:control group,VPA group(15?M VPA treatment for 24h alone),HPTA group(15?M HPTA treatment for 24h alone),HU group(2mM treatment for 18h alone),HU+VPA group(15?M VPA pretreatment 24h after combined with 2mM treatment for 18h),HU+HPTA group(15?M HPTA pretreatment 24h after combined with 2mM treatment for 18h).After the drug treatment was completed,the absorbance values of each group were detected at the wavelengths of 490nm&532nm of the microplate reader,and the average value was taken.Compared the average value of each group,the average value reflects the proliferation of breast cancer cells.2.Cell clone formation assay to detect the effect of drugs on breast cancer cell survivalMCF7 and EUFA423 cells in logarithmic growth phase were seeded in p60 culture dishes to divided into six groups respectively:control group,VPA group(500?M VPA treatment for 24h alone),HPTA group(15?M HPTA treatment for 24h alone),HU group(2mM treatment for 18h alone),HU+VPA group(500?M VPA pretreatment 24h after combined with 2mM treatment for 18h),HU+HPTA group(15?M HPTA pretreatment 24h after combined with 2mM treatment for 18h).After the treatment,the fresh DMEM medium(containing 10%GST+1%PS)was replaced,and the cells were allowed to culture for 14 days in the incubator.Cell clones were formed at the bottom of the petri dish,and the culture was terminated to calculate the clone formation rate which reflected the proliferation ability of the cells.3.The detection of the nuclear DNA DSBsComet assay experiment:select breast cancer cells in logarithmic growth phase,cell grouping:control group,HPTA group(15?M HPTA treatment for 24h alone),HU group(2mM treatment for 18h alone),HU+HPTA group(15?M HPTA pretreatment 24h after combined with 2mM treatment for 18h),after the drug treatment finished,the cells of each group were collected to make a single cell suspension,and then neutral comet and alkaline comet experiments according to manufacturing instruction were performed to observe the cell tailing.The DNA damage in the nucleus analyzed.Immunofluorescence and Western blot experiments:After the drug treatment,the cells of each group were tested for the formation of foci and protein expression of yH2AX and 53BP1 by immunofluorescence and immunoblotting experiments.The expression of ?yH2AX,53BP1 foci and protein in each group reflects the differences in DNA DSBs.4.Flow cytometry was used to detect the effect of HPTA on HR repair efficiency of cells.The cells of MCF7/pDR-GFP were selected in the logarithmic growth phase and treated with HPTA(15uM HPTA treatment for 24h alone),HU(2mM treatment for 18h alone),HU+HPTA(15uM HPTA pretreatment 24h after combined with 2mM treatment for 18h).After the drug treatment was completed,the treated cells were collected to make into a single cell suspension,and the effect of the drug on the HR repair efficiency of each group of cells was detected by flow cytometry.5.The detection of HR repair mechanism key proteins RPA2-p and Rad51.The cells in the logarithmic growth phase were selected and treated with HPTA(15?M HPTA treatment for 24h alone),HU(2mM treatment for 18h alone),HU+HPTA(15?M HPTA pretreatment 24h after combined with 2mM treatment for 18h).After the drug treatment,the expressions of RPA2-p and Rad51 proteins in each group of cells were detected by immunoblotting and immunofluorescence experiments.6.Detection of the interaction between RPA2-p and Rad51Co-Immunoprecipitation(Co-IP)was used to study whether the relationship between RPA2p and Rad51 protein coexisted in the same protein complex.7.Establishment of a pair of MCF7 cell lines stably expressing wild-type RPA2 and mutant RPA2The exogenous wild-type RPA2 protein was mutated,and the mutated RPA2 could not normally perform the phosphorylation function of the protein,and the exogenous mutant RPA2 was obtained.Then,exogenous wild-type and mutant RPA2 were transfected into normal MCF7 cells,respectively,and shRPA2 was used to knock out endogenous RPA2 of the cells.Cell cultures stably expressing wild-type RPA2 and mutant RPA2 were then selected.8.Obtaining primary cultured cells from rat primary breast cancerDissolve dimethylbenzanthrene(DMBA)powder in a small amount of toluene and then dissolve it in vegetable oil.50-day-old sexually mature SD female rats were orally administered with 20mg DMBA orally to rats.SD rats were raised for approximately 90 days and induced with primary breast tumors.Under aseptic conditions,rat primary breast tumors were removed and HE stained to identify the character of the tumor.Another part of the tumor tissue was shredded and then subjected to primary cell culture for related experiments.Results1.HPTA at low doses can make breast tumor cells sensitive to HU treatmentThe results of MTT experiments further showed that 15?M HPTA alone treatment inhibited the proliferation of MCF7 cells(P<0.05),while 15?MVPA alone had no significant effect on cell proliferation(P>0.05).Compared with the HU treatment group alone,15?M HPTA combined with HU can further improve the killing ability of tumor cells(P<0.05),but 15?M VPA cannot(P>0.05).EUFA423 cells MTT test data also confirmed that 15?M HPTA can further improve the killing ability of HU to tumor cells,and 15?M VPA cannot significantly improve the killing effect of HU on tumor cells.The results of the breast cancer cell line MCF7 clone formation assay showed that compared with the control group,the survival fraction of cells treated with 500?M VPA or 15?M HPTA for 24 h was reduced to 78.28%(P>0.05)or 67.72%(P>0.05).The survival fraction of cells treated with 2mM HU for 18 h decreased to 37.49%(P<0.01).After 24h of 500?M VPA or 15?M HPTA pretreatment and further combination treatment with HU,the survival fraction was decrease to 11.72%(P<0.01)and 19.37%.The survival fraction of the two combined treatment groups showed similar patterns of change,and there was no statistical difference,indicating that the combination of HPTA and HU had a combined killing effect on MCF7 tumor cells.Moreover,it is suggested that 15?M HPTA can increase the sensitivity of breast cancer cells to HU as well as 500?M VPA.The results of the breast cancer cell line EUFA423 clone formation assay showed that compared with the control group,the survival fraction of cells treated with 2mM HU for 18 h was decrease to 61.43%(P<0.01).For 500?M VPA or 15?M HPTA combined with HU,the survival fraction decreased to 51.60%or 55.34%,respectively.It shows that 15?M HPTA and 500?M VPA have similar effects on increasing the sensitivity of breast tumor cells to HU.The above results further confirm that low-dose HPTA can increase the sensitivity of breast tumor cells to HU and suggest that it may be more efficient than VPA.2.HPTA at low doses can enhance HU-induced DNA double-strand break damage in breast cancer cellsThe results of both neutral comet and alkaline comet assay showed that compared with the control group,the cell DNA tailing of the cells HPTA-treated group increased slightly,and the cell DNA tailing distance of the HU-treated group increased significantly.The combination of HPTA and HU further increased the cell DNA tailing,which was the longest among the four groups(P<0.05).Immunofluorescence results showed that MCF7 cells contained a small amount of ?H2AX foci in the control group.The positive rate in the HPTA group increased to 13.07%(P<0.05),and in the HU-only group it increased significantly to 35.1%(P<0.05).while,for the cells treated with HPTA combined with HU,the positive rate of cells with yH2AX foci increased to 52.27%(P<0.01).In EUFA423 cells,compared to the control group,15?M HPTA caused an increase in foci formation of yH2AX.In the HPTA and HU combined treatment group,the positive rate of cells with yH2AX foci formation was higher than in the other groups(P<0.01).Similar results were observed for another marker of DNA DSBs,53BP1 protein.In MCF7 and EUFA423 cells,after treatment with HU,the positive rate of cells containing 53BP1 foci increased(P<0.01).HPTA and HU combined treatment group,the positive rate of cells containing the 53BP1 foci increased further significantly(P<0.01).Immunoblotting results showed that in MCF7 and EUFA423 cells,the expression of yH2AX protein in control cells was detected to be low,and only a small amount of DNA DSB was accumulated in MCF7 cells treated with HPTA.After HU treatment,the expression of ?H2AX in cells increased significantly.On this basis,the combination of HPTA and HU can further increase the expression of yH2AX protein in cells significantly(P<0.01).And in MCF7 and EUFA423 cells,the expression level of 53BP1 protein in combined group was also significantly higher than that in the single treatment group(P<0.01).3.HPTA inhibits HR activity after HU-induced DNA replication arrestMCF7/PDR-GFP cells were used to detect the efficiency of HR repair in each group by flow cytometry.The results showed that the HR repair efficiency of the control group was 60 × 10-6,in the HPTA-treated group,the HR repair efficiency was 65×10-6(P>0.05).However,after HU treatment,the HR repair efficiency was significantly increased to 100×10-6(P<0.01).The combined effect of HPTA and HU on HR repair efficiency was significantly reduced to 66×10-6 compared with the effect of HU alone(P<0.01),indicating that HPTA can inhibit the HR repair function of HU-induced replication fork block in MCF7cells.4.HPTA inhibited the function of RPA2-p and Rad51 in HU-induced DNA replication block repair.The expression of RPA2-p protein in MCF7 and EUFA423 cells in each group was detected by immunobloting.We found that HPTA treatment alone increased the expression intensity of RPA2-p protein slightly(MCF7:0.42;EUFA423:0.32);HU group increased the expression intensity of RPA2-p protein significantly(MCF7:0.53;EUFA423:0.44);HPTA and HU combined treatment significantly reduced the intensity of RPA2-p protein expression(MCF7:0.22;EUFA423:0.33).The same change trend was also obtained in each treatment group of the two cells by immunofluorescence experiment.Immunoblotting experiments detected the expression level of Rad51 protein in each group.The results showed that HU alone treatment in MCF7 cells only increased the expression level of Rad51 protein slightly(1.53),and the combined effect of HPTA and HU decreased the expression level of Rad51 protein(1.25,P<0.01).In EUFA423 cells,compared with the HU-treated group alone(0.90),the combined effect of HPTA and HU decreased the expression level of Rad51 protein(0.78;P<0.05).The results of further immunofluorescence experiments showed that in MCF7 cells,compared with the control group,the positive rate of containing Rad51 foci cells in the HU-treated group significantly increased to 33.45%(P<0.05).Compared with the HU-treated group alone,HPTA and HU combined group,the positive rate of Rad51-containing foci cells decreased significantly to 19.25%(P<0.01).The same change trend was also obtained in each treatment group in EUFA423 cells.5.Interaction between RPA2-p and Rad51,the key proteins in HU-induced DNA replication block repairThe results of co-immunoprecipitation(Co-IP)experiments showed that two proteins,RPA2-p and Rad51,existed in the same protein complex precipitate and there was an interaction between the two proteins.6.Effects of HPTA on survival of the cells expressing defective RPA2-p after HU treated.The results of western blotting showed that the expression of RPA2-p protein in mutRPA2 cells was significantly reduced compared to wtRPA2 cells after HU treatment,indicating that a mutant RPA2-p cell line was successfully established.The results of the colony formation experiments showed that the HPTA treatment group did not significantly reduce the survival fraction of the paired cells(P>0.05).After HU treatment alone(P<0.01),the survival fraction of wt-RPA2 cells was reduced to 38.75%,and the survival fraction of mutRPA2 cells was decreased to 22.89%.Compared with wt-RPA2 cells,mutRPA2 cells were more sensitive to HU treatment(P<0.01).In the combined HPTA and HU group,the survival fraction of wt-RPA2 cells was reduced to 18.20%,and the survival rate of mutRPA2 cells was decreased to 13.60%.There was no significant difference in survival fraction between the two cells(P>0.05).The above results further confirm that the reason why HPTA can cause cell death that it disrupted the RPA2-p-attended HR repair pathway.7.Effect of HPTA and HU on DMBA-induced primary breast cancer cells in ratsThe tumor tissue was obtained as sections,and the pathological structure of the tissue was observed by hematoxylin-eosin(HE)staining.After HE staining,it was confirmed as rat breast cancer tissue.Immunofluorescence experiments detected the percentage of cells containing yH2AX foci in the nucleus.The results showed that there were fewer cells with yH2AX foci formation in the control group,HPTA treatment alone and HU-treated group slightly increased the percentage of cells with yH2AX foci formation to 33.87%,and the combined application of 2mM HU and 15?M HPTA significantly increased the percentage of cells with yH2AX foci formation to 43.98%(P<0.01).Immunofluorescence experiments were performed to detect the positive rates of the repair proteins RPA2-p and Rad51.The results showed that the HU-treated group significantly increased the positive rate of cells expressing RPA2-p foci to 40.50%when compared with the control group,while the percentage of cells expressing RPA2-p foci in the HPTA and HU combined group was significantly reduced to 34.87%(P<0.05).In addition,compared with the control group,the expression of Rad51 foci in the nucleus of cells significantly increased the percentage of cells expressing Rad51 foci in the HU treatment group to 32.30%.The percentage of cells expressing Rad51 foci in the combined HPTA and HU group significantly decreased to 27.00%(P<0.05).Conclusions1.By the working system of two tumor cell lines and the primary culture cells obtained from DMBA-induced rat breast cancer tissue,it was first discovered that low-dose 15?M HPTA had the similar effect for the enhancement of HU-induced DNA double-strand break damage and increase the sensitivity of HU to kill breast cancer cells as compared with 500?M VPA,indicating that HPTA may be more effective than VPA for killing tumor cells.2.Low-dose HPTA inhibits RPA2-p-Rad51-mediated HR pathway to increase the sensitivity of breast cancer cells to HU. |
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