Abstract：
This study explores the specific role and underlying mechanisms of ALDH5A1 in the chemoresistance of esophageal squamous cell carcinoma (ESCC). The levels of cleaved caspase-3, 4-hydroxynonenal (4-HNE), intracellular Fe2+, and lipid reactive oxygen species (ROS) were evaluated via immunofluorescence. Cell viability and migration were quantified using cell counting kit-8 assays and wound healing assays, respectively. Flow cytometry was utilized to analyze cell apoptosis and ROS production. The concentrations of malondialdehyde (MDA) and reduced glutathione were determined by enzyme-linked immunosorbent assay. Proteome profiling was performed using data-independent acquisition. Additionally, a xenograft mouse model of ESCC was established to investigate the relationship between ALDH5A1 expression and the cisplatin (DDP)-resistance mechanism in vivo. ALDH5A1 is overexpressed in both ESCC patients and ESCC/DDP cells. Silencing of ALDH5A1 significantly enhances the inhibitory effects of DDP treatment on the viability and migration of KYSE30/DDP and KYSE150/DDP cells and promotes apoptosis. Furthermore, it intensifies DDP\'s suppressive effects on tumor volume and weight in nude mice. Gene ontology biological process analysis has shown that ferroptosis plays a crucial role in both KYSE30/DDP cells and KYSE30/DDP cells transfected with si-ALDH5A1. Our in vitro and in vivo experiments demonstrate that DDP treatment promotes the accumulation of ROS, lipid ROS, MDA, LPO, and intracellular Fe2+ content, increases the levels of proteins that promote ferroptosis (ACSL4 and FTH1), and decreases the expression of anti-ferroptosis proteins (SLC7A11, FTL, and GPX4). Silencing of ALDH5A1 further amplifies the regulatory effects of DDP both in vitro and in vivo. ALDH5A1 potentially acts as an oncogene in ESCC chemoresistance. Silencing of ALDH5A1 can reduce DDP resistance in ESCC through promoting ferroptosis signaling pathways. These findings suggest a promising strategy for the treatment of ESCC in clinical practice.
摘要：
本研究探讨ALDH5A1在食管鳞状细胞癌化疗耐药中的作用及其机制。裂解的caspase-3，4-羟基壬烯醛（4-HNE）的水平，胞内Fe2+,和脂质活性氧（ROS）通过免疫荧光进行评估。使用细胞计数试剂盒-8测定和伤口愈合测定对细胞活力和迁移进行定量。分别。流式细胞术用于分析细胞凋亡和ROS产生。用酶联免疫吸附法测定丙二醛（MDA）和还原型谷胱甘肽的浓度。使用独立于数据的采集进行蛋白质组分析。此外,建立ESCC异种移植小鼠模型，研究ALDH5A1表达与体内顺铂（DDP）耐药机制之间的关系。ALDH5A1在ESCC患者和ESCC/DDP细胞中均过表达。ALDH5A1的沉默显著增强DDP处理对KYSE30/DDP和KYSE150/DDP细胞活力和迁移的抑制作用,促进细胞凋亡。此外，它增强了DDP对裸鼠肿瘤体积和重量的抑制作用。基因本体论生物学过程分析表明，铁凋亡在转染si-ALDH5A1的KYSE30/DDP细胞和KYSE30/DDP细胞中起着至关重要的作用。我们的体外和体内实验表明，DDP处理促进ROS的积累，脂质ROS，MDA,LPO,和细胞内Fe2+含量，增加促进铁凋亡的蛋白质（ACSL4和FTH1）的水平，并降低抗铁凋亡蛋白的表达(SLC7A11，FTL，和GPX4）。ALDH5A1的沉默进一步放大了DDP在体外和体内的调节作用。ALDH5A1可能在ESCC化疗耐药中充当癌基因。沉默ALDH5A1可通过促进铁凋亡信号通路降低ESCC对DDP的耐药性。这些发现为临床实践中ESCC的治疗提供了有希望的策略。
