S. M. Bai1,2, X. B. Wan1, X. J. Fan1,3, and Y. L. Wang1; 1Department of Radiation Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China, 2GuangDong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China, 3Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
Purpose/Objective(s): Radiation-induced intestinal injury has a significant impact on the effectiveness of radiotherapy and the quality of life for patients undergoing pelvic radiotherapy. Our goal is to offer novel theoretical support and identify new therapeutic targets for the prevention and treatment of radiation-induced intestinal injury. Materials/
Methods: To investigate the mechanism underlying radiosensitivity, we constructed a radioresistant HeLa cell line. By conducting RNA-seq analysis, we compared the mRNA expression profiles of the radiation-resistant HeLa cells and the wild-type Hela cells. Among the differential genes, we identified proteins related to the endocytosis pathway. For mechanistic research, CRISPR/cas9 was used to construct cell lines of gene-knocking out. Then, we used flow cytometry, a cell counting kit, colony formation, and Western blotting to investigate the relationship between endocytosis and Radiation-induced intestinal injury. Results: According to the KEGG pathways analysis of RNA-seq data, the endocytosis pathway was significantly enriched from differentially expressed genes between radiation-resistant cell line and parental radiation-sensitive cell line. Interestingly, colorectal cancer (CRC) samples with low expression of CAV1, an important mediator of endocytosis, showed higher radioresistance. Consistently, pitstop2, an inhibitor of clathrin-dependent and independent endocytosis, was confirmed to enhance radioprotection in both cells and mice model of radiation-induced intestinal injury. Interestingly, we found that Pitstop 2 had no impact on radiation-induced DNA damage repair and cell apoptosis, but significantly inhibited ferroptosis. Further investigations revealed that the increase of ferroptosis post-irradiation was mainly caused by the decrease of Cystine/glutamate antiporter SLC7A11 which could be rescued by pitstop2 and BafA1. Additionally, SLC7A11 and RAB7 showed colocalization post irradiation, suggesting that SLC7A11 was down-regulated by endocytosis and lysosome-mediated degradation. Importantly, the clinically available drug prochlorperazine (PCZ), an inhibitor of dynamin-mediated endocytosis, could also improve cell viability after irradiation, suggesting that targeting SLC7A11 endocytosis may be a potential strategy for the prevention and treatment of radiation-induced intestinal injury. Conclusion: After irradiation, SLC7A11 was degraded through CAV1-mediated endocytosis and the lysosomal pathway, thereby promoting ferroptosis of intestinal cells. Pitstop2 and the clinically available drug PCZ has the potential as an effective protective drug for radiation-induced intestinal injury.
