Effects Of LDR On Tumor Inhibition Of HDR And Its DNA Damage Repair Mechanism

As a conventional tumor treatment,radiotherapy is one of the most commonly used methods of tumor treatment in clinical practice.However,its radiotherapy efficacy is affected by the tumor microenvironment and tumor cell radioresistance after the action of ionizing radiation,so that the tumor cannot be completely cured.Therefore,how to improve the radiosensitivity of tumor cells and improve the efficacy of clinical tumor radiotherapy has become a research hot topic in the field of radiation oncology and radiobiology.Background:A large amount of scientific information has demonstrated the value and potential of the hormesis of low dose radiation(LDR)in the treatment of malignant tumors,and it mainly works through activation of immune intercellular responses and T-cell signaling pathways,promotion of cytotoxic t lymphocytes(CTL)and natural killer cells(NK),and upregulation of cytotoxic effects such as tumor necrosis factor-α(TNF-α)and gamma-interfering cells(NK).The LDR mainly promotes cytotoxic t lymphocyte(CTL)and natural killer cell(NK)cytotoxic effects,and upregulates tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)cytokine expression,thus it can enhance the body’s anti-tumor immune function and improving tumor The immunosuppressive state caused by tumor load.LDR refers to low LET radiation at doses less than 0.2 Gy or high LET radiation at doses less than 0.05 Gy.LDR can cause various biological effects,such as adaptive responses,genomic instability,hypersensitivity and bystander effect.Animal experiments and a few clinical case studies have reported that the combination of LDR(in the form of whole-body or half-body irradiation)with radiotherapy and chemotherapy can improve tumor treatment effects,inhibit tumor metastasis and improve radiotherapy efficacy,which has important potential value in tumor treatment.However,studies on the tumor suppressive effect of LDR local irradiation modality on enhanced high dose radiation(HDR)have not been reported.Ionizing radiation kills tumor cells mainly works by inducing double-strand breakage(DSB),while tumor cells are capable of DSB repair to varying degrees.There are two repair pathways in human cells:non-homologous end joining(NHEJ)repair based on DNA-dependent protein kinase(DNA-PK)complexes,and homologous recombination(HR)repair based on recombination protein A(Rad51).Recent studies have shown that Rad51 and DNA-PKcs protein expression levels are associated with different radiation responses in tumor cells.In this study,we used in vitro cellular experiments and vivo animal experiments to investigate the effect of LDR on the tumor suppressive effect of HDR and its DNA damage repair mechanism,in order to provide a reliable theoretical basis for the application of LDR in clinical tumor radiotherapy.Objective:In this study,we used a co-culture model(tumor cells/immune cells)to detect the expression of genes and proteins related to DNA damage repair in vitro,and also used an immunodeficient mouse tumor model to observe the proliferation inhibition,immunogenic death and related cytokine secretion,the extent of DNA damage and its repair in HDR-induced lung cancer cells,and further elaborate the molecular mechanism of DNA damage repair in the process,aiming to provide a new theoretical basis for improving the efficacy of clinical tumor radiotherapy.Methods:1.A co-culture model of A549(lung adenocarcinoma)cells and Jurkat(in vitro T lymphocyte model)cells was established and divided into Control group,LDR group,LDR+HDR group and HDR group.2.The CCK8 method was used to detect changes in the proliferation rate of A549cells after irradiation in the co-culture model.3.Changes in apoptosis rate of A549 cells in co-culture model after irradiation were detected by flow cytometry using Annexin V and PI apoptosis kit staining.4.Flow cytometry was used to detect changes in the cell cycle progression of A549cells after irradiation in the co-culture model.5.The immunofluorescence method was used to detect the changes in the number ofγH2AX fluorescent foci and the number of 53BP1 repair foci in A549 cells after irradiation of the co-culture model.6.The m RNA expression of two pathways related molecules of DNA damage repair were d by q RT-PCR.7.A tumor-bearing nude mouse model was established,and the changes in tumor volume were recorded in mice for 21 days after irradiation.8.Extracted tumor-bearing tissues and m RNA expression of DNA damage repair pathway-related molecules were detected by q RT-PCR.9.Western Blot method was used to detect the expression of DNA damage repair pathway and pyroptosis related molecular protein in cell co-culture model.10.Irradiation conditions:In vitro cell irradiation conditions were LDR dose of100 m Gy,dose rate of 13.4 m Gy/min,HDR dose of 20 Gy,dose rate of 1.02 Gy/min;in vivo experimental irradiation used local irradiation,LDR dose of 100 m Gy,the dose rate was 13.4 m Gy/min,the HDR dose was 20 Gy,and the dose rate was 1.98 Gy/min.11.Statistical treatment:statistical analysis was performed using SPSS 24.0software,and the results of the obtained experimental data were expressed as mean±standard deviation(`x±s).Statistical graphs were drawn using Graph Pad Prism8software.Statistical analysis was performed using two independent samples t-test,and differences were considered statistically significant if p<0.05.Results:1.Establish a co-culture model of A549 cells and Jurkat cellsA549 cells were walled cells,and Jurkat cells were suspension cells.After A549cells were cultured to the exponential growth stage,they were inoculated into cell culture dishes(plates),and after A549 cells were walled,Jurkat cells at the exponential growth stage were inoculated into cell culture dishes(plates).After the co-culture cells were stabilized,the cells were prepared for irradiation treatment,and the interval between LDR pre-irradiation and HDR irradiation was 6 h.2.Effect of LDR on HDR inhibition of A549 cell proliferation in the tumor/immune cell co-culture modelThe results of CCK8 showed that after the co-culture model was successively irradiated with two doses of X-rays,compared with the Control group,the cell proliferation rate in the LDR+HDR group and the HDR group decreased significantly,the LDR group decreased slightly,and the cell proliferation rate in the LDR+HDR group was significantly lower than that in the HDR group.However,LDR did not enhance the proliferation inhibitory effect of HDR in A549 cultured cells alone.It is suggested that LDR can enhance the inhibitory effect of HDR on the proliferation of A549 cells in a co-culture model.3.Effects of LDR on HDR-induced apoptosis and pyroptosis of A549 cells in a co-culture modelThe changes of apoptosis was detected by flow cytometry.The results showed that the apoptosis rate was slightly increased in the LDR group and significantly increased in the LDR+HDR and HDR groups compared with the control group,and the apoptosis rate in the LDR+HDR group was significantly higher than that in the HDR group(p<0.05).This result indicates that LDR pre-irradiation could enhance the killing effect of HDR on tumor cells under the co-culture conditions of A549 cells and Jurkat cells.The protein expressions of pyroptosis-related molecules NLRP3,Cleaved-caspase-1 and GSDMD were detected by Western Blot.The results showed that the expression of pyroptosis-related proteins in the LDR+HDR group and the HDR group was higher than that in the control group,and the expression in the LDR+HDR group was higher than that in the HDR group.This indicated that pyroptosis occurred in the co-culture model after irradiation,and LDR pre-irradiation could enhance HDR-induced pyroptosis.4.Effect of LDR on HDR-induced A549 cell cycle distribution in a co-culture modelFlow cytometry was used to detect the cell cycle progression.That means a significant G2 phase block occurred in the LDR+HDR and HDR groups compared with the control group,and the G2 phase block was more pronounced in the LDR+HDR group than in the HDR group.It is suggested that giving HDR after pre-irradiation of LDR can increase the percentage of cells in the G2 phase,the most sensitive cycle of radiation,and thus improve radiosensitivity.5.Effects of LDR on HDR-induced DNA damage repair in A549 cells in a co-culture modelThe protein expression of DNA damage markerγH2AX and DNA repair marker53BP1 was detected by immunofluorescence assay,and the results showed that the number ofγH2AX foci was significantly increased in the LDR+HDR group and HDR group compared with the control group,and the number ofγH2AX foci was significantly more in the LDR+HDR group compared with the HDR group,while in the results of 53BP1 detection,compared with the control group,the number of 53BP1foci was increased in the LDR+HDR and HDR groups,and the number of 53BP1 foci in the LDR+HDR group was significantly less than that in the HDR group significantly.It indicated that LDR enhanced HDR-induced DNA damage in A549 cells under co-culture conditions,while inhibiting DNA damage repair.The m RNA expression of Rad51 and BRCA1,markers of HR repair pathway,and DNA-PKcs,XRCC4 and Ku80,markers of NHEJ repair pathway,were detected in A549 cells 48 h after irradiation in co-culture model by q RT-PCR.The results revealed that the m RNA expression of the above five genes was significantly reduced in both the LDR+HDR and HDR groups compared with the control group;the m RNA expression of the HR repair pathway-related molecules Rad51 and BRCA1 was reduced in the LDR+HDR group compared with the HDR group(p<0.05,p<0.01),and similarly,the NHEJ repair pathway-related molecules Ku80,DNA-PKcs and XRCC4 m RNA expression were significantly reduced in the HDR group compared to the HDR group(p<0.05,p<0.01,p<0.01).Western Blot was used to detect the expression of HR and NHEJ repair pathway markers BRCA1,Rad51,DNA-PKcs,XRCC4 and Ku80 proteins in A549 cells 72 h after irradiation in the co-culture model.The results showed that the protein expression of all five molecules was reduced in both the LDR+HDR and HDR groups compared with the Control group,and the protein expression of the five molecules was significantly lower in the LDR+HDR group than in the HDR group.It showed that LDR-enhanced killing effect of HDR on tumor cells was related to two DNA damage repair pathways under co-culture conditions.6.Observing the effect of LDR on the tumor suppressive effect of HDR at the overall level using a nude mouse tumor-bearing model(1)Establishment of nude mouse tumor-bearing modelTwenty female BALB/CA-nu nude mice aged 5 weeks and weighing 18-22 g were selected and randomly divided into Control group,LDR group,LDR+HDR group and HDR group,with 5 mice in each group.After the nude mice were stabilized,1×10~6A549 cells were injected into their right leg,and after the injection,the nude mice were observed and waited for a mass of about 4 mm~3 to grow on the right leg,indicating the successful establishment of the lotus tumor model.(2)Changes in tumor volume of tumor-bearing nude mice after irradiationWhen the tumors grew to about 215 mm~3,they were randomly divided into Control group,LDR group,LDR+HDR group and HDR group.The tumor size was measured every 3 days after irradiation,and the nude mice were executed and tumors were peeled off after 21 days of irradiation.The results showed that the tumor volume in the LDR+HDR group was smaller than that in the HDR group(p<0.05),and the tumor volume in the LDR,LDR+HDR and HDR groups was smaller than that in the control group(p<0.05),indicating that LDR could enhance the radiotherapeutic efficacy of HDR on tumors.It can be seen that LDR local irradiation significantly enhanced the tumor suppressive effect of HDR.7.DNA damage repair mechanism of LDR-enhanced HDR radiotherapy efficacy in a tumor-bearing nude mouse modelThe m RNA expression of DNA damage-repair-related genes in nude mice hormonal tumor tissues was detected by q RT-PCR.The results showed that the m RNA expression levels of HR and NHEJ repair pathway-related molecules Rad51,BRCA1,DNA-PKcs,XRCC4 and Ku80 were significantly reduced in the LDR+HDR group compared with the control group(p<0.01),and Rad51 and Ku80 m RNA expression were reduced in the LDR+HDR group compared with the HDR group(p<0.05).BRCA1,DNA-PKcs and XRCC4 m RNA expression were significantly reduced compared to HDR group(p<0.01).It indicates that the enhanced tumor suppressive effect of HDR by LDR pre-irradiation is achieved by inhibiting its DNA damage repair.Conclusions:1.When individual tumor cells were irradiated,the tumor suppressive effect of LDR on HDR was not enhanced.2.When lung cancer cells coexist with immune cells in vitro,LDR enhances the proliferation inhibitory effect of HDR on lung cancer cells by promoting the G2 phase block of lung cancer cells.3.When lung cancer cells coexist with immune cells in vitro,LDR enhances the tumor suppressive effect of HDR by inducing pyroptosis and apoptosis of lung cancer cells.4.In in vivo animal experiments,LDR local pre-irradiation can inhibit DNA damage repair,which in turn enhances the tumor suppressive effect of HDR local irradiation.