背景:缺氧在鼻咽癌化疗耐药中起重要作用。铁凋亡是一种新发现的程序性细胞死亡形式,铁凋亡诱导剂在某些癌症中显示出有希望的治疗效果。然而,缺氧微环境下NPC细胞对铁凋亡的敏感性尚不清楚,这项研究旨在澄清这一点。
方法:NPC细胞,用Erastin治疗,置于37℃的常氧或低氧环境(5%CO2,94%N2和1%O2)中24h。通过CCK8、MDA、GSH,脂质ROS和Fe。从TCGA数据库下载头颈部鳞状细胞癌(HNSCC)组织的基因表达谱,筛选构建分子。筛选出BAP1,并通过敲低BAP1检测其对擦除素诱导的NPC细胞铁凋亡的功能。进行荧光素酶报告基因测定和co-IP实验以探索分子机制。最后,应用肿瘤异种移植模型在体内进一步验证这些结果。
结果:CCK8实验表明,在缺氧条件下,用erasin处理的NPC细胞的IC50显着低于常氧下的IC50。低氧显著增加脂质ROS和MDA的水平,并降低了erastin诱导的GSH含量。基于TCGA数据库构建并验证了具有6个铁凋亡相关基因的HNSCC的预后风险模型。缺氧条件下BAP1显著上调,荧光素酶报告基因分析表明HIF-1α是BAP1的上游转录调节因子。NPC细胞中BAP1的敲除在缺氧下显着增加了erastin的IC50值,并在脂质ROS方面显着改善了erastin在缺氧下诱导的铁细胞凋亡。MDA含量和GSH。Co-IP成果显示,BAP1介导H2A的去泛素化和SLC7A11的表达下降。最后,在肿瘤异种移植模型中,BAP1的敲低降低了对擦除素诱导的铁凋亡的敏感性。而BAP1敲低细胞移植瘤中H2A程度显著下降。
结论:缺氧诱导的BAP1通过稳定H2A增强了Erastin诱导的NPC铁凋亡。靶向BAP1的Ferroptosis诱导剂可能是改善鼻咽癌化疗耐药的有效途径,尤其是在缺氧的微环境中。
BACKGROUND: Hypoxia plays an important role in the chemotherapy resistance of nasopharyngeal carcinoma (NPC). Ferroptosis is a newly discovered form of programmed cell death and ferroptosis inducers showed promising therapeutic effects in some cancers. However, the sensibility of NPC cells to ferroptosis under the hypoxic microenvironment is still unclear, and this study was designed to clarify it.
METHODS: NPC cells, treated with erastin, were placed in a normoxia or hypoxic environment (5% CO2, 94% N2 and 1% O2) at 37℃for 24 h. After exposed to hypoxia, ferroptosis-associated phenotypes were detected by CCK8, MDA, GSH, lipid ROS and Fe. The gene expression profiles of head and neck squamous cell carcinoma (HNSCC) tissues were downloaded from the TCGA database to screen construction molecule.
BAP1 was screened out and its functions on erastin-induced ferroptosis in NPC cells were detected by knockdown of
BAP1. Luciferase reporter assay and co-IP experiment were performed to explore the molecular mechanism. Finally, the tumour xenograft model was applied to further verify these results in vivo.
RESULTS: CCK8 assay showed that IC50 of NPC cells treated with erastin under hypoxia was significantly lower than that under normoxia. Hypoxia significantly increased the levels of lipid ROS and MDA, and decreased GSH content induced by erastin. A prognostic risk model for HNSCC with six ferroptosis-related genes was constructed and validated based on TCGA database. BAP1 was significantly up-regulated under hypoxia, and luciferase reporter assay showed that HIF-1α was an upstream transcription regulator of
BAP1. Knockdown of BAP1 in NPC cells significantly increased the IC50 value of erastin under hypoxia and significantly ameliorated erastin-induced ferroptosis under hypoxia in aspect of lipid ROS, MDA content and GSH. Co-IP results showed that
BAP1 mediated deubiquitination of H2A and decreased SLC7A11 expression. Finally, knockdown of BAP1 reduced sensitivity to erastin-induced ferroptosis in a tumour xenograft model. And the level of H2A was significantly decreased in xenograft tumors of
BAP1 knockdown cells.
CONCLUSIONS: Hypoxia-induced BAP1 enhances erastin-induced ferroptosis in NPC by stabilizing H2A. Ferroptosis inducers targeting BAP1 may be an effective way to improve chemotherapy resistance in NPC, especially in the hypoxic microenvironment.