| BackgroundCurrently,radiotherapy（RT）is a common clinical treatment for breast cancer.Because of the radiation resistance and systemic adverse reactions during RT treatment to tumor,the efficacy of RT will be greatly reduced.Therefore,finding the safe and effective radiosensitizers,which can not only increase the sensitivity of RT,but also effectively avoid the systemic adverse reactions in the process of RT treatment has become a major topic in the field of tumor radiotherapy.2-hexyl-4-pentynoic acid（HPTA）is a derivative of the antiepileptic drug valproate（VP A）,both of which are VPA-like compounds and belong to the histone deacetylase inhibitors（HDACi）.The IC50 of HPTA for inhibition of HD AC activity is only 11-15μM,which is much lower than that of VPA（348-448μM）,suggesting that HPTA may be a more efficient alternative to VPA.Our latest study showed that VPA has radiosensitizing effect on breast cancer cells,and found that VPA can target ubiquitin ligase RAWD3 to promote recombinase Rad51 ubiquitination,reduce Rad51 protein expression and inhibit homologous recombination（HR）mechanism of DNA double-strand breaks（DSBs）damage repair to achieve radiosensitization to tumor cells.But whether HPTA has a radiosensitizing effect on breast cancer cells,the possible sensitizing mechanism,and replace VPA to become a safer and efficient sensitizer have not been reported so far.There are two mechanisms of DNA DSBs damage repair,HR and Nonhomologous end joining（NHEJ）,in which HR is an accurate error-free form,and thus has attracted much attention.Rad51 is a key enzyme in HR repair and its functional status can directly affect DNA damage repair as well as genome stability.It has been reported that Rad51 expression levels are regulated not only by the ubiquitin ligase RAWD3 but also by deubiquitinating enzymes（DUBs）during DNA damage repair,which coordinately regulate the level of Rad51,thereby maintaining it in a stable state to adapt to the needs of the organism.UCHL3,a member of the deubiquitinating enzyme family,can directly deubiquitinate Rad51 and promote BRCA2-Rad51 binding and recruitment of Rad51,thus participating in the DNA damage repair process.This suggests that the HR mechanism regulated by UCHL3-Rad51 pathway is also important in maintaining the functional state of the cell.However,whether HPTA can target the UCHL3-Rad51 deubiquitination pathway to influence the effect of IR on cancer cells has not been reported.Notably,radiotherapy can also cause tumor immune tolerance,leading to tumor progression and recurrence,and then affect the sensitivity of radiotherapy,so how to reverse the tumor immune tolerance caused by radiotherapy or activate the anti-tumor immune response to increase the sensitivity of tumor cells to radiation,is also a bottleneck that needs to be broke through in the tumor radiotherapy field.Recently,it has been demonstrated that HDACi can achieve tumor suppressive effects by increasing the expression of immunogenic molecules in tumors,and VPA can decrease immune escape to inhibit tumor growth by inhibiting HDACs.Whether HPTA can reverse radiotherapy induced tumor immune tolerance by activating the anti-tumor immune response remains unclear.Based on the above studies,we put forward the following hypotheses:HPTA may act as a more efficient and safer radiosensitizer compared to VPA;It may target the UCHL3-Rad51 deubiquitination pathway to regulate HR mechanism and increase radiosensitivity of breast cancer cells to achieve significant anti-tumor efficacy;Meanwhile,HPTA may also reverse tumor immune escape and enhance radiosensitization to tumor cells by activating anti-tumor immune response mechanisms.Aiming at discovering multi-target radiosensitizing formulations that can inhibit DNA damage repair and activate anti-tumor immune response,our study will provide a reliable theoretical and experimental basis for the application of HPTA in clinical cancer radiotherapy.Chapter Ⅰ The study on radiosensitizing effect of HPTA to breast cancer cellsObjects:Using VPA as a positive control,through breast cancer cells,environmental carcinogen（DMBA）-induced transformation of MCF10A normal mammary epithelial cells（MCF10A-DMBA）established by our group and primary breast cancer rat model,this chapter of the study systematically explored the radiosensitization effect of low concentration HPTA on breast cancer cells as well as the effect of HPTA on IR induced damage of normal tissues and cells,at both cellular and animal level.Methods:1.Detection of the effect of HPTA on radiosensitivity of breast tumor cellsBreast cancer cell lines as well asMCF10A-DMBA cells were applied.The dose of HPTA treated cells was first detected.For the treated cells,the radiosensitization effect and concentration of HPTA,cell survival was investigated by MTT assay and coloney formation assay,respectively.2.Detection of the effect of HPTA on radiosensitivity of primary breast cancer rat modelApplying the primary breast cancer rat model established by our group,we first detected the dose of HPTA treated animals and the manner in which HPTA was combined with IR.Ten days after treatment,the following assays were performed:measurement and recording of changes in tumor size,H＆E staining to observe pathological and morphological changes,immunohistochemistry to detect the amount of cells staining for proliferation markers BrdU and Ki67 and for apoptosis markers cleaved caspase-3,immunohistofluorescence to assess the expression of CD31,an endothelial cell surface marker,in tissue vessels.3.Detection of the effect of HPTA on IR induced-damage of normal tissue cellsFor the treated MCF10A cells,cell survival was detected by coloney formation assay.For the treated primary breast cancer rat model,the body weight changes and the organ index of major viscera（spleen,liver,lung,and brain）as well as the amount of proliferative cells in spleen and small intestine staining for BrdU were detected.Results:1.Radiosensitization effect of low dose HPTA on breast cancer cellsFirst,we determined that the radiosensitizing dose of HPTA was 15μM.It was found that 15μM HPTA combined with IR significantly inhibited MCF7 cell growth,which was similar to the effect of 500μM VPA combined with IR;15μM HPTA can effectively increase the sensitivity of MCF7 and MCF10A-DMBA cells to IR and decrease the cell survival.2.Radiosensitization effect of HPTA on primary breast cancer rat modelIt was first determined to employ fractionated low-dose IR（2 Gy/fraction/day for 4 consecutive days）for orthotopic tumor radiation,as well as to select 20 mg/kg HPTA for the treatment in a combined manner in which HPTA treatment was administered throughout the IR treatment.It was found that tumor volume decreased more significantly in the combination group than in the IR group;More and larger vacuolated necrotic structures appeared within the tumor tissues of the combination group;The number of cells expressing the cell proliferation markers BrdU and Ki67 was significantly decreased in the tumor tissues after the combination treatment,but the number of cells expressing the apoptosis marker cleaved caspase-3 was significantly increased;the CD31（a surface marker of endothelial cells）positive region within the tumor tissue（the density of blood vessels）were significantly reduced,suggesting that the proliferative capacity of tumor cells was reduced,apoptosis was increased,and vessel growth was inhibited.3.Effect of HPT A on IR induced damage of normal tissue cellsAt the cellular level,15μM HPTA did not show radiosensitization of MCF10A.In the animal model,orthotopic tumor IR leads to a significant decrease in body weight,but 20 mg/kg HPTA restores body weight to normal levels,the organ index of liver and spleen were recovered,the number of cells with proliferative capacity was also significantly increased in spleen and small intestine.Conclusion:1.HPTA is a highly efficient radiosensitizer of tumor tissue cells,using breast cancer cell models,it was found that 15μM HPTA exerts radiosensitizing effects on cancer cells;using a primary breast tumor model,we found that 20 mg/kg HPTA exerts radiosensitizing effects on tumor tissues.2.HPTA is a safe radiosensitizer for tumor tissue cells:while exerting its radiosensitizing effect on cancer cells,HPTA also attenuates IR induced damage to normal tissue cells.Chapter Ⅱ HPTA targets the UCHL3-Rad51-mediated HR pathway to increase radiosensitization of breast cancerObject:Throughout the first chapter,using multiple breast cancer cell models and primary breast cancer rat model,it was found that low concentration of HPTA had similar radiosensitizing effects with high dose of VP A,suggesting that the IR-induced DNA DSBs repair mechanisms,HR and NHEJ,may be involved in the radiosensitization of cancer cells by HPTA.In this chapter,we will apply the above models to explore the radiosensitization mechanism of HPTA.Methods:1.Detection of the effect of HPTA on IR induced DNA DSBs within breast cancer cellsFor the treated MCF7 as well as MCF10A-DMBA cells,the intracellular DSBs damage,DSBs damage markers γH2AX and 53BP1 foci formation and protein expression were investigated by neutral and alkaline comet assay,cellular immunofluorescence assay and western blotting,respectively.For the primary breast cancer rat model,immunohistochemistry and western blotting to detect yH2AX protein expression in the tumor tissues as well as cellular immunofluorescence assay to detect γH2AX and 53BP1 foci formation in primary cultured tumor cells.2.Detection of the effect of HPTA on HR mechanism of IR induced DNA DSBsFor MCF7 cells expressing the pDR-GFP recombination reporter,transfected with I-SceI nuclease to inflict intracellular DNA DSBs,and cells with GFP expression were assessed by flow cytometry to assess HR efficiency;Flow cytometry was used to detect the cell cycle changes of MCF7 cells after treatment;Foci formation,protein expression and mRNA expression of Rad51 and BRCA1 were detected by cellular immunofluorescence assay,western blotting and real-time PCR,respectively;The clonogenic ability of cells after treatment was examined in the cells which BRCA1-deficiency or Rad51-inhibited.3.Detection of the effect of HPTA on NHEJ mechanism of IR induced DNA DSBsFor U2OS cells expressing the EJ5-GFP recombination reporter,transfected with I-SceI nuclease to inflict intracellular DNA DSBs,and cells with GFP expression were assessed by flow cytometry to assess NHEJ efficiency;The protein levels of the key proteins（DNA-PKCs,Ku70,and Ku80）were detected by western blotting.4.Detection of the effect of HPTA on Rad51 stability and ubiquitinationThe half-lives of BRCA1 and Rad51 proteins in MCF7 cells treated with cycloheximide were detected by western blotting;The protein level of Rad51 after MG 132 treatment was detected by western blotting;Co-IP assay was performed to detect Rad51 protein ubiquitination.5.Detection of the effect of HPTA on the UCHL3-Rad51 mediated deubiquitination pathwayUCHL3 protein level was detected by western blotting;Co-IP assay was performed to detect the interaction between UCHL3 and Rad51;After downregulating UCHL3 expression by siRNA,Rad51 protein level and ubiquitination level were detected by western blotting and CO-IP assay,respectively.6.Detection of the mechanism of radiosensitization by HPTA in primary breast cancer rat modelThe Rad51 protein level was detected by immunohistochemistry and western blotting;The UCHL3 protein level was detected by western blotting.Results:1.Effect of HPTA on IR induced DNA DSBs within breast cancer cellsAt the cellular level,HPTA aggravated IR induced DSBs damage in MCF7 cells;In both MCF7 as well as MCF10A-DMBA cells,HPTA significantly increased IR induced γH2AX and 53BP1 foci formation and protein expression.At the animal level,a significant increase in yH2AX protein expression appeared within the tumor tissues of the combination group.For the primary cultured tumor cells,yH2AX and 53BP1 foci formation was significantly increased after combined treatment compared with IR treatment alone.2.Effect of HPTA on HR mechanism of DNA DSBs15μM HPTA significantly reduced HR efficiency by 42.88%,and HPTA had no effect on the cell cycle;Compared with the IR group,15μM HPTA significantly reduced foci formation and protein levels of Rad5 1 and BRCA1 in both MCF7 and MCF10A-DMBA cells;Both BRCA1 deficiency and Rad51 downregulation significantly reduced HPTA radiosensitization effect.3.Effect of HPTA on NHEJ mechanism of DNA DSBs15μM HPTA significantly reduced NHEJ efficiency by 19.48%,but HPTA had no significant effect on the main proteins（DNA-PKCs,Ku70 and Ku80）involved in NHEJ repair.4.Relationship between HPTA induced radiosensitization and Rad51 protein stability as well as protein ubiquitinationAfter cycloheximide treatment of MCF7 cells,HPTA combined with IR significantly shortened the half-life of BRCA1 and Rad51;The reduction of Rad51 protein level induced by HPTA combined with IR was reversed by treatment with MG132;Co-IP assay further revealed that HPTA combined with IR significantly increased the ubiquitination level of Rad51.5.Relationship between HPTA induced radiosensitization of cancer cells and the UCHL3-Rad51 deubiquitination pathwayAt the cellular level,the combination of HPTA and IR significantly suppressed UCHL3 protein levels;There is an interaction between UCHL3 and Rad51;After down-regulating UCHL3 expression by siRNA,the decrease of Rad51 protein level was reversed after combined treatment.At the animal level,using the primary breast cancer rat model,both Rad51 and UCHL3 protein levels were significantly reduced after combined treatment compared with IR treatment alone;The number of cells expressing Rad51 within tumor tissues was significantly decreased after combined treatment compared with IR treatment alone.Conclusion:Using breast tumor cells and rat primary breast tumor models,at both cellular and animal levels,we found that HPTA can increase the accumulation of IR-induced DNA DSBs,target the deubiquitinating enzyme UCHL3 to regulate Rad51 ubiquitination level,inhibit Rad51 function,downregulate the Rad51 mediated HR pathway and increase radiosensitization of cancer cells.Chapter Ⅲ HPTA radiosensitizes cancer cells through activating anti-tumor immune responseObject:Based on the previous two chapters,it was demonstrated that HPTA was a highly effective and safe radiosensitizer at the cellular and animal levels,and targeted UCHL3-Rad51 deubiquitination pathway to exert radiosensitization.In recent years,tumor progression and recurrence caused by RT-induced tumor immune tolerance have also attracted increasing attention and it is also unknown whether the enhanced efficacy of RT by HPTA is related to its regulation of immune function.Therefore,in this chapter,primary breast cancer rat model as well as co-culture models were applied to explore whether HPTA could activate immune cells（macrophages as well as cytotoxic CD8+T cells）dependent anti-tumor immune functions and exert radiosensitizing effect on cancer cells at both animal and cellular levels.Methods:1.Detection of the relationship between anti-tumor immune activation and macrophage polarization by HPTAPrimary breast cancer rat model was applied,for the tumor samples on the 10th day post-treatment:Immunohistochemistry was performed to detect the positive expression of macrophage markers F4/80 and CD68 in each group;Real time PCR was used to detect the markers of macrophage classification and their secreted cytokines in tumor tissues.Immunohistochemistry and immunohistochemistry were performed to identify the origin of macrophages and their phagocytic ability.At the cellular level,macrophages were treated with conditioned medium incubated with MCF7 to examine the effect of HPTA on macrophage polarization,and macrophage classification markers and their secreted cytokines were detected by real-time PCR and ELISA;MTT assay was performed to examine the effect of HPTA treated macrophage lysate on the growth of irradiated MCF7 cells.2.Detection of the relationship between anti-tumor immune activation and CD8+ T cells by HPTAPrimary breast cancer rat model was applied,for the tumor samples on the 10th day post-treatment:immunohistochemistry was performed to evaluate the positive expression of CD8+T cells and granzyme B in each group.At the cellular level,cell lysates from macrophages RAW264.7 treated with HPTA were applied to activated mononuclear cells（PBMCs）isolated from the peripheral blood of healthy individuals,and CD8+ T cell numbers were assessed by flow cytometry;PBMCs containing activated CD8+T cells were further co-cultured with irradiated MCF7 cells to test MCF7 growth by MTT assay.3.Detection of sustained long-term efficacy of anti-tumor immune response by HPTAPrimary breast cancer rat model was applied,for the tumor samples on the 70th day post-treatment,the following tests were performed,respectively:Measurement and recording of changes in tumor size,H＆E staining to observe pathological and morphological changes,immunohistochemistry to detect the amount of cells staining for proliferation markers BrdU and Ki67;Macrophage polarization and activated CD8+ T cells were detected by immunohistochemistry,real-time PCR,and immunohistochemistry;immunohistofluorescence to assess the expression of CD31 in tissue vessels.Results:1.Effect of the anti-tumor immune response mediated by HPTA activated M1 macrophages on radiosensitization of cancer cellsAt the animal level,compared with IR treatment alone,the number of F4/80 and CD68 positive cells were significantly increased in tumor tissues after combined treatment,in which M1 macrophage markers（CD86 and MHC-Ⅱ）and the related cytokines（IFN-γ、IL-12、IL-6 and TNF-α）were significantly increased,but M2 macrophage markers（CD209 and CD 163）and the related cytokine（IL-10）were significantly decreased,suggesting that the radiosensitizing effect of HPTA may be related to M1 macrophage infiltration,illustrating that HPTA may promote macrophage reprogramming;Within the tumor tissues,the number of CD11b positive cells was significantly increased after HPTA combined with IR treatment,while EpCAM and F4/80,markers of breast tumor cells,showed better immunofluorescence colocalization,which deeply revealed that the infiltrated macrophages were from the myeloid lineage and had strong phagocytosis.At the cellular level,when RAW264.7 macrophages were treated with conditioned medium of cultured tumor cells,it was found that HPTA could induce the macrophages to polarize to M1 type,which showed the increase of M1 markers and IL-12 cytokines,and the decrease of M2 markers and IL-10 cytokines,which was consistent with the results of animal level.Then,irradiated MCF7 cells were treated with cell lysates from HPTA activated Ml macrophages,and the findings indicated that the growth of irradiated tumor cells was further inhibited,thereby suggesting that M1 macrophage anti-tumor immune pathways may be involved in the radiosensitization of cancer cells by HPTA.2.Effect of the anti-tumor immune response mediated by HPTA activated macrophage-CD8+T cells on radiosensitization of cancer cellsAt the animal level,a significant increase in the number of CD8+T cells and granzyme B,a marker of activated CD8+T cell were observed in combination treatment,suggesting that the radiosensitizing effect of HPTA may be related to the infiltration of activated CD8+T cells within the tumor tissue.At the cellular level,cell lysates from M1 macrophages activated with HPTA were applied to PBMCs,and flow cytometry results showed that CD8+ T cell were significantly increased in PBMCs,suggesting that HPTA can activate CD8+T cells through activated macrophages.Then,co-culture of PBMCs containing activated CD8+ T cells with irradiated MCF7 cells revealed that the growth of irradiated cells was also significantly inhibited,suggesting that the M1 macrophage-CD8+T cell antitumor immune pathway may be involved in the radiosensitization of cancer cells by HPTA.3.Sustained long-term efficacy of anti-tumor immune response by HPTAAt the animal level,at 70 days post-treatment,the combined treatment still significantly inhibited tumor growth,a large number of necrotic areas appeared in the tumor tissue,significantly reduced proliferative capacity of tumor cells,sparse blood vessel density in the tumor tissue,and massive infiltration of M1 macrophages and CD8+ T cells within the tumor tissue.The above results suggest that immune cellmediated responses activated by HPTA can persist and maintain their strong antitumor efficacy.Conclusion:1.HPTA is a potent immune activator and not only activates M1 macrophage mediated anti-tumor immune pathway but also macrophage--CD8+T cell mediated anti-tumor immune pathway,all of which are involved in the radiosensitization of cancer cells by HPTA.2.Immune functions mediated by HPTA activated immune cells can persist and maintain strong anti-tumor immune efficacy. |
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