Enhancement of the Tumor Suppression Effect of High-dose Radiation by Low-dose Pre-radiation Through Inhibition of DNA Damage Repair and Increased Pyroptosis

Abstract

Radiation therapy has been a critical and effective treatment for cancer. However, not all cells are destroyed by radiation due to the presence of tumor cell radioresistance. In the current study, we investigated the effect of low-dose radiation (LDR) on the tumor suppressive effect of high-dose radiation (HDR) and its mechanism from the perspective of tumor cell death mode and DNA damage repair, aiming to provide a foundation for improving the efficacy of clinical tumor radiotherapy. We found that LDR pre-irradiation strengthened the HDR-inhibited A549 cell proliferation, HDR-induced apoptosis, and G2 phase cell cycle arrest under co-culture conditions. RNA-sequencing showed that differentially expressed genes after irradiation contained pyroptosis-related genes and DNA damage repair related genes. By detecting pyroptosis-related proteins, we found that LDR could enhance HDR-induced pyroptosis. Furthermore, under co-culture conditions, LDR pre-irradiation enhances the HDR-induced DNA damage and further suppresses the DNA damage-repairing process, which eventually leads to cell death. Lastly, we established a tumor-bearing mouse model and further demonstrated that LDR local pre-irradiation could enhance the cancer suppressive effect of HDR. To summarize, our study proved that LDR pre-irradiation enhances the tumor-killing function of HDR when cancer cells and immune cells were coexisting.

Keywords: DNA damage repair; high-dose radiation; inflammatory cytokines; low-dose radiation; pyroptosis; tumor suppression effect.

Figure 1.

There is no effect of LDR on the tumor suppression effect of HDR when irradiating tumor cells alone. (A) Cell viability of A549 cells after irradiation was evaluated by using CCK8 assay, n = 4 per group. (B–D) Cell viability of tumor cells after irradiation was evaluated by using CCK8 assay, n = 5 per group. (E–G) The apoptosis rate of tumor cells after irradiation was detected by using flow cytometry, n = 3 per group. All data are presented as the mean ± SD.

Figure 2.

LDR could enhance the inhibitory effect of HDR on tumor cells under co-culture conditions. (A) Co-culture model was established by using A549 cells and Jurkat cells. (B) Cell viability of A549 cells after co-culture cell irradiation was evaluated by using CCK8 assay, n = 5 per group. (C, D) The apoptosis rate of A549 cells after co-culture cell irradiation was detected by using flow cytometry, n = 3 per group. (E, F) Flow cytometry analysis of the proportion of the different cell cycle phases in A549 cells after co-culture irradiation, n = 3 per group. All data are presented as the mean ± SD. *P < .05, **P < .01.

Figure 3.

Pyroptosis and DNA damage repair may be potential mechanisms. (A) The Venn diagram shows the number of differential genes. (B–D) The volcano plot shows the number of genes whose expression was upregulated or downregulated in the LDR group, LDR+HDR group, and HDR group compared to the Control group. (E) Clustering of genes altered in the Control group, LDR group, LDR+HDR group, and HDR group. (F–H) Gene ontology analysis of differentially expressed genes in the LDR group, LDR+HDR group, and HDR group compared to the Control group. (I–K) KEGG was used for pathway analysis of differential genes.

Figure 4.

LDR pre-irradiation could enhance HDR-induced pyroptosis under co-culture conditions. (A) NLRP3, GSDMD, and cleaved-caspase-1 protein levels in A549 cells were determined after 48 h of irradiation. (B-E) Expressions of IL-18, IL-1β, TNF-α, and IL-32 in the supernatant after irradiation, n = 3 per group. (F) The expression of NLRP3 after transfection with siRNA targeting NLRP3 in A549 cells was assayed by using Western blot technique. (G) The expression of NLRP3 was assayed after treating A549 cells with 10 μM NLRP3 inhibitor-MCC950 by using Western blot technique. (H) A549 cells were transfected with si-NLRP3 and then co-cultured with Jurkat cells, cell survival was detected by using CCK8 assay after irradiation, n = 4 per group. (I) A549 cells were treated with MCC950 and then co-cultured with Jurkat cells, and cell survival was detected with CCK8 after irradiation, n = 4 per group. (J) A549 cells were transfected with si-NLRP3 and then co-cultured with Jurkat cells, and pyroptosis-related proteins were assayed by using Western blot technique after irradiation. (K) A549 cells were treated with MCC950 and then co-cultured with Jurkat cells, and pyroptosis-related proteins were assayed by using Western blot technique after irradiation. All data are presented as the mean ± SD. *P < .05, **P < .01.

Figure 5.

LDR pre-irradiation enhances DNA damage caused by HDR irradiation and inhibits DNA damage repair. (A) Immunofluorescence staining for γH2AX in A549 cells after 3 h of irradiation. Scale bars: 40 μm. (B) The number of focal points of γH2AX, n = 25 per group. (C) Immunofluorescence staining for 53BP1 in A549 cells after 3 h of irradiation. Scale bars: 50 μm. (D) The number of focal points of 53BP1, n = 20 per group. (E-I) Relative expressions of Rad51, BRCA1, Ku80, DNA-PKcs, and XRCC4 mRNA after 48 h of irradiation, n = 3 per group. (J) Rad51, BRCA1, Ku80, DNA-PKcs, and XRCC4 protein levels in A549 cells were determined after 72 h of irradiation. All data are presented as the mean ± SD. *P < .05, **P < .01.

Figure 6.

LDR no longer enhances the inhibitory effect of HDR on tumor cells after treatment of VND3207. (A) A549 cells were treated with VND3207(40 μM) for 2 h and co-cultured with Jurkat cells, and after irradiation the survival rate of A549 cells was assessed by using CCK8 assay, n = 3 per group. (B, C) A549 cells were co-cultured with Jurkat cells after VND3207 treatment, and the apoptosis rate of A549 cells was detected by using flow cytometry after irradiation, n = 3 per group. (D–H) Relative expressions of Rad51, BRCA1, Ku80, DNA-PKcs, and XRCC4 mRNA after 48 h of irradiation, n = 3 per group. (I) Ku80, DNA-PKcs, and XRCC4 protein levels in A549 cells were determined after 72 h of irradiation. All data are presented as the mean ± SD. *P < .05, **P < .01.

Figure 7.

LDR local irradiation enhances the tumor suppressive effect of HDR in tumor-bearing mice. (A) Tumor location in the tumor-bearing mouse. (B) Cells were injected subcutaneously into the right leg of nude mice. When the tumor volume reached approximately 200 mm³, the mice were treated with irradiation. The tumors were excised 21 days after irradiation. (C) The average volume of the tumors was measured every 3 days after irradiation. (D-H) Relative expressions of Rad51, BRCA1, Ku80, DNA-PKcs, and XRCC4 mRNA in tumor tissues. (I) The protein levels of NLRP3, GSDMD, cleaved-caspase-1, Rad51 and XRCC4 were measured in tumor tissues. All data are presented as the mean ± SD, n = 4. *P < .05, **P < .01.

Figure 8.

Illustration of the mechanism by which LDR enhances the tumor-suppressive effect of HDR. When tumor cells coexist with immune cells, LDR pre-irradiation increases HDR-induced G2 phase cell cycle arrest, increases HDR-induced DNA damage, and inhibits DNA damage repair after HDR irradiation, causing an increase in HDR-induced apoptosis, meanwhile, radiation leads to the secretion of some pro-inflammatory factors, causing the onset of pyroptosis, and LDR pre-irradiation can enhance HDR-induced pyroptosis, ultimately leading to proliferation inhibition of tumor cells.