褪黑素通过诱导铁死亡抑制食管鳞癌生长的机制研究

目的 探讨褪黑素通过抑制溶质运载蛋白7家族成员11（SLC7A11）表达对食管鳞癌细胞铁死亡的影响。方法 使用不同浓度（0.25、0.5、1.0、2.0、4.0 mmol/L）的褪黑素处理人食管鳞癌KYSE150细胞，筛选建立KYSE150细胞铁死亡模型的褪黑素浓度；将KYSE150细胞分为对照组、褪黑素（1.0 mmol/L）组、褪黑素（1.0 mmol/L）＋pcDNA（转染空载质粒）组、褪黑素（1.0 mmol/L）＋SLC7A11（转染SLC7A11过表达质粒）组。透射电子显微镜观察KYSE150细胞线粒体形态；碘化丙啶（PI）染色检测细胞死亡；试剂盒检测细胞Fe²⁺、谷胱甘肽（GSH）及活性氧（ROS）水平；Western blotting法检测细胞SLC7A11蛋白表达。建立食管鳞癌裸鼠荷瘤模型，将BALB/c裸鼠分为模型组、褪黑素（ip 25 mg/kg褪黑素）组、褪黑素＋pcDNA（ip 25 mg/kg褪黑素＋裸鼠右侧腋下注射转染空载质粒的KYSE150细胞）组、褪黑素＋SLC7A11（ip 25 mg/kg褪黑素＋裸鼠右侧腋下注射转染SLC7A11过表达质粒的KYSE150细胞）组；分别检测各组小鼠肿瘤体积、质量及肿瘤组织中SLC7A11蛋白表达。结果 1.0 mmol/L褪黑素可诱导KYSE150细胞铁死亡；与对照组比较，褪黑素组KYSE150细胞死亡数目增多，细胞内的线粒体变小，线粒体膜密度增加，细胞内Fe²⁺及ROS水平显著增加（P＜0.05），SLC7A11蛋白表达水平及细胞内GSH水平显著降低（P＜0.05）；与褪黑素＋pcDNA组比较，褪黑素＋SLC7A11组细胞死亡数目减少，细胞内的线粒体增大，线粒体膜密度减少，细胞内Fe²⁺及ROS水平显著降低（P＜0.05），SLC7A11蛋白表达水平及细胞内GSH水平显著升高（P＜0.05）；褪黑素可通过抑制SLC7A11蛋白表达抑制食管鳞癌裸鼠肿瘤生长。结论 褪黑素可能通过抑制SLC7A11表达诱导细胞铁死亡，抑制食管鳞癌细胞生长。

Mechanism of melatonin inhibiting the growth of esophageal squamous cell carcinoma by inducing iron death

Objective To investigate the effect of melatonin on ferroptosis of esophageal squamous cell carcinoma by inhibiting the expression of solute carrier protein 7 family member 11 (SLC7A11). Methods The KYSE150 cells were treated with different concentrations of melatonin (0.25, 0.5, 1.0, 2.0, 4.0 mmol/L), and the concentrations of melatonin were screened to establish the iron death model of KYSE150 cells. KYSE150 cells were divided into control group, melatonin (1.0 mmol/L) group, melatonin (1.0 mmol/L) + pcDNA (transfected with empty plasmid) group, and melatonin (1.0 mmol/L) + SLC7A11 (transfected with SLC7A11 overexpression plasmid) group. Mitochondrial morphology of KYSE150 cells was observed by transmission electron microscope. The cell death was detected by propyl iodide (PI) staining. The levels of Fe²⁺, glutathione (GSH), and reactive oxygen species (ROS) were detected by the kit. The expression of SLC7A11 protein was detected by Western blotting. Establishing a tumor bearing model of esophageal squamous cell carcinoma in nude mice. BALB/c nude mice were divided into model group, melatonin (ip 25mg/kg melatonin) group, melatonin + pcDNA (ip 25mg/kg melatonin + KYSE150 cells transfected with empty plasmids injected into the right armpit of nude mice) group, and melatonin +SLC7A11 (ip 25mg/kg melatonin + KYSE150 cells transfected with SLC7A11 overexpression plasmid by right armpit injection of nude mice) group. Tumor volume, mass and expression of SLC7A11 protein in tumor tissues were detected. Results 1.0 mmol/L melatonin was able to induce ferroptosis of KYSE150 cells. Compared with control group, the death number of KYSE150 cells in melatonin group increased, intracellular mitochondria became smaller, mitochondrial membrane density increased, intracellular Fe²⁺ and ROS levels were significantly increased (P < 0.05), SLC7A11 protein expression level and intracellular GSH level were significantly decreased (P < 0.05). Compared with melatonin + pcDNA group, the number of cell death in melatonin + SLC7A11 group was decreased, intracellular mitochondria were increased, mitochondrial membrane density was decreased, intracellular Fe²⁺ and ROS levels were significantly decreased (P < 0.05), SLC7A11 protein expression level and intracellular GSH level were significantly increased (P < 0.05). Melatonin can inhibit the tumor growth of nude mice with esophageal squamous cell carcinoma by inhibiting SLC7A11 protein expression. Conclusion Melatonin may induce cell ferroptosis and inhibit the growth of esophageal squamous cell carcinoma by inhibiting the expression of SLC7A11.