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

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.

The transcription factor GATA3 positively regulates NRP1 to promote radiation-induced pulmonary fibrosis.

Radiation-Induced Pulmonary Fibrosis (RIPF) frequently arises as a delayed complication following radiation therapy for thoracic cancers, encompassing lung, breast, and esophageal malignancies. Characterized by a relentless and irreversible accumulation of extracellular matrix (ECM) proteins within the lung parenchyma, RIPF presents a significant clinical challenge. While the modulation of gene expression by transcription factors is a recognized aspect in various pathologies, their specific role in the context of RIPF has been less clear. This study elucidates that ionizing radiation prompts the translocation of the transcription factor GATA3 into the nucleus. This translocation facilitates GATA3's binding to the NRP1 promoter, thereby enhancing the transcription and subsequent translation of NRP1. Further investigations demonstrate that the TGF-ß pathway agonist, SRI-011381, can mitigate the effects of NRP1 knockdown on epithelial-mesenchymal transition (EMT) and ECM deposition, suggesting a pivotal role of the GATA3/NRP1/TGF-ß axis in the pathogenesis of RIPF. In conclusion, our findings not only underscore the critical involvement of GATA3 in RIPF but also highlight the GATA3/NRP1/TGF-ß signaling pathway as a promising target for therapeutic intervention in RIPF management.

AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy.

BACKGROUND: Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma. METHODS: The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. RESULTS: Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. CONCLUSIONS: This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.

Epigenetic regulation in radiation-induced pulmonary fibrosis.

PURPOSE: Radiation-induced pulmonary fibrosis (RIPF) is a common and serious adverse effect of radiotherapy for thoracic tumors, which occurs in the irreversible stage of radiation-induced lung injury (RILI) >6 months after irradiation. It is characterized by progressive and irreversible destruction of lung tissue and deterioration of lung function, which may impair quality of life and lead to respiratory failure and death. We hope this will draw attention to the involvement of epigenetics in the regulation of RIPF. CONCLUSIONS: This review summarizes research progress on the role and mechanism of DNA methylation, noncoding RNA and RNA methylation in RIPF or RILI, and the possible role and mechanism of histone modification in RIPF. We have noticed that in tissue fibrosis, the epigenetic regulation mechanisms inside and outside the nucleus can influence each other. We speculate that RIPF may be regulated by an epigenetic regulatory network during its development, and believe that TGF-ß, SNAIL, PTEN and EZH2 are four targets worthy of in-depth study.

GATA3 Exerts Distinct Transcriptional Functions to Regulate Radiation Resistance in A549 and H1299 Cells.

Background: Radiation resistance of lung cancer cells is a vital factor affecting the curative effect of lung cancer. Transcription factor GATA3 is involved in cell proliferation, invasion, and migration and is significantly expressed in a variety of malignancies. However, the molecular mechanism governing GATA3 regulation in lung cancer cells' radiation resistance is unknown. Methods: Radiation-resistant cell models (A549-RR and H1299-RR) were made using fractionated high-dose irradiation. Use clone formation, CCK-8, F-actin staining, cell cycle detection, and other experiments to verify whether the model is successfully constructed. Cells were transiently transfected with knockdown or overexpression plasmid. To explore the relationship between GATA3/H3K4me3 and target genes, we used ChIP-qPCR, ChIP-seq, and dual luciferase reporter gene experiments. Xenograft tumor models were used to evaluate the effect of GATA3 depletion on the tumorigenic behavior of lung cancer cells. Results: We report that transcription factors GATA3 and H3K4me3 coactivate NRP1 gene transcription when A549 cells develop radiation resistance. However, the mechanism of radiation resistance in H1299 cells is that GATA3 acts as a transcription inhibitor. The decrease of GATA3 will promote the increase of NRP1 transcription, in which H3K4me3 does not play a leading role. Conclusions: GATA3, an upstream transcriptional regulator of NRP1 gene, regulates the radioresistance of A549 and H1299 cells by opposite mechanisms, which provides a new target for radiotherapy of lung cancer.

Effect of EG00229 on Radiation Resistance of Lung Adenocarcinoma Cells.

Background: Neuropilin 1 (NRP1) is a pleiotropic receptor that interacts with multiple ligands and their receptors and plays a critical role in the process of tumor metastasis and radiation resistance in endothelial cells and tumor cells. In this study, we sought to investigate the mechanistic role of NRP1 in the radiation resistance of non-small cell lung cancer (NSCLC) cells and the role of EG00229 (an inhibitor of NRP1) on reversing radiation resistance. Materials and Methods: A549 and H1299 NSCLC cells were used to construct radiation resistance models. Western blot, ELISA, and qRT-PCR were used to detect protein and mRNA levels of NRP1, epithelial-mesenchymal transition (EMT) markers, and molecules in signaling pathways. Immunofluorescence was used to measure changes in co-expression of NRP1 and VEGF-165 in radiation-resistant model cells. An immunoprecipitation assay was used to detect the binding capacity of NRP1 and VEGF-165. Results: We successfully created two radiation resistant models (A549RR and H1299-RR). The expression levels of NRP1, EMT-related proteins, and proteins in metastasis-related pathways were increased in NSCLC cells with radiation resistance. After adding EG00229, the expression levels and binding capacity of NRP1 and VEGF-165 proteins were significantly reduced. The expression of EMT-related proteins and proteins in metastasis-related pathways were reduced in NSCLC cells with radiation resistance. Conclusion: Our data provide an insight into the molecular mechanisms of radiation resistance and suggest that EG00229 may contribute to reversing the radiation resistance of NSCLC cells by inhibiting the binding of NRP1 and VEGF-165. Our findings could provide a novel theoretical and experimental foundation for improving the efficacy of lung cancer radiotherapy.

NRP1 regulates radiation-induced EMT via TGF-ß/Smad signaling in lung adenocarcinoma cells.

PURPOSE: Radiation has been shown to promote the epithelial-mesenchymal transition (EMT) in tumor cells, and TGF-ß/Smad and PI3K-Akt signaling pathways play an important role in the EMT. In this study, we investigated the effects of neuropilin-1 (NRP1) on radiation-induced TGF-ß/Smad and non-classical Smad signaling pathways in lung cancer cells, as well as the effects of NRP1 on invasion and migration. MATERIALS AND METHODS: Changes in the expression levels of EMT markers (ß-catenin, N-cadherin, and vimentin) and related transcription factors (Twist and ZEB1) in stably transfected cells were detected by Western blotting and qPCR, and changes were assessed by TGF-ß/Smad and non-classical Smad signaling. Immunofluorescence was used to detect the expression of the cytoskeletal protein F-actin. Expression of TGF-ß1 and CXCL-12 was detected by ELISA. Transwell and scratch assays were used to detect the invasive ability and migration of lung cancer cells, respectively. RESULTS: Our results showed that ionizing radiation could induce the EMT as well as morphological changes in lung adenocarcinoma cells (A549); however, the effects were not significant in lung squamous carcinoma cells (SK-MES-1). Moreover, we showed that NRP1 promotes the EMT induced by ionizing radiation in A549 cells, which may be related to the increased expression of EMT-related transcription factors. NRP1 may promote the radiation-induced EMT of A549 cells mainly through TGF-ß1/Smad2/3 signaling. NRP1 also enhanced radiation-induced invasion, migration, and CXCL-12 expression in A549 cells. CONCLUSIONS: We conclude that NRP1 promotes radiation-induced EMT in lung adenocarcinoma cells via TGF-ß1/Smad signaling and not non-classical Smad signaling, and enhances the invasion and migration of lung adenocarcinoma cells.

The Role of the Tumor Microenvironment in Neuropilin 1-Induced Radiation Resistance in Lung Cancer Cells.

Background: Neuropilin 1 (NRP1) is a pleiotropic receptor which can interact with multiple ligands and their receptors. It plays an important role in the process of axonal growth, angiogenesis, tumor metastasis and radiation resistance in endothelial cells and some tumor cells. Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis, and has received considerable attention in recent years. Material and Methods: In this study, A549 lung cancer cell lines with different NRP1 expression levels were constructed in vitro, a two-dimensional (2D), three-dimensional (3D) co-culture system and tumor-bearing model was established in SCID mice. Western blot, qRT-PCR, immunofluorescence, cytometric bead array and flow cytometry were used to investigate the effect of the tumor microenvironment in NRP1-induced lung cancer cell radiation resistance. Results: In 2D or 3D co-culture system, NRP1 could be regulated inflammatory factors such as TNF, IL-6 IL-8 and IL-17 and the related chemokines MCP-1, IP-10 and RANTES in the tumor microenvironment, which in turn induced radiation resistance in lung cancer cells. In addition, different expression levels of NRP1 in 2D, 3D culture systems and tumor-bearing models were able to significantly regulate cell phenotype, proliferative capacity, epithelial-mesenchymal transition (EMT) and the radiation resistance of A549 cells. Conclusion: Our results verified that NRP1, inflammatory factors, chemokines and related signaling pathways, which affect the transformation of related cell components and thus lung cancer cell immune tolerance and migratory ability, all play an important role in radiation resistance.

Mechanism of MEN1 gene in radiation-induced pulmonary fibrosis in mice.

OBJECTIVE: To investigate the regulatory mechanism of MEN1 gene in radiation-induced lung fibrosis in mice and provide a new theoretical basis for the clinical treatment of radiation pulmonary fibrosis. METHODS: First, 80 C57BL/6 mice aged 8 weeks and weighing 18-22 g were selected, half of them were male and the other half were female. The mice were divided into control group and irradiation group (40 mice in each group) according to the method of the random number table. A radiation-induced lung fibrosis mouse model was established in which a single X-ray irradiation of 20 Gy was applied to the right lung in the irradiation group; H&E and Masson staining were used to verify whether the model was successful at 4, 8, 16 and 24 weeks after irradiation. The expression of MEN1, smooth muscle actin (α-SMA), Collagen-1 and transforming growth factor (TGF-ß) in lung tissue were detected by Western blot and qPCR. Secondly, in the mouse embryonic fibroblast cell line (MEF) and mouse lung epithelial cell line (MLE-12), we constructed cell models of MEN1 knockout and interference separately with the irradiation of 10 Gy X-rays. The expression of α-SMA, Collagen-1, and TGF-ß/Smads signaling pathway molecules was detected by qPCR. Finally, using the immunoprecipitation (IP) method, we can detect the interaction between Smad2 and the protein menin encoded by the MEN1 gene. RESULTS: The results of the radiation pulmonary fibrosis model in mice showed that compared with the control group, the alveolar septum widens, the alveolar integrity decreases, the lung tissue slightly thickens, and a small amount of collagen deposits appear after 4-8 weeks in the model group. At twenty-fourth weeks, a large number of cells in the interstitial space of the lung tissue and a localized focal fibrosis area were observed. Further study found that radiation induced fibrogenic inflammatory cytokines TGF-ß up-regulation, down-regulation of MEN1 gene expression, and then enhanced the expression of α-SMA and promotes the transformation of fibroblasts to myofibroblasts; At the same time, the expression of Collagen-1 was enhanced, which suggested that the extracellular matrix was overconcentrated and eventually promoted the formation of pulmonary fibrosis. In vitro, we found that knockout and interference of MEN1 gene can significantly enhance radiation-induced fibrosis, and up-regulate the expression of downstream molecules Smad2 and Smad3 of TGF-ß signaling pathway, and down-regulate the expression of Smad7. Furthermore, it played an important role in regulating the process of radionuclide fibrosis. CONCLUSION: MEN1 plays a key role in the formation of pulmonary fibrosis by regulating the secretion of TGF-ß and the activation of TGF-ß/Smads signaling pathway.