摘要
目的探索紫草素(SKN)抑制甲状腺未分化癌(ATC)细胞生长的作用及分子机制。方法以流式细胞仪检测SKN对ATC细胞系CAL-62细胞铁死亡的影响;蛋白质印记法检测NF-κB、铁死亡相关基因谷胱甘肽过氧化物酶4(GPX4)和硒蛋白硫氧还蛋白还原酶(TXNRD1)、糖代谢相关基因丙酮酸激酶同工酶2(PKM2)和葡萄糖转运蛋白(GLUT1)的表达水平变化;实时荧光定量PCR检测GPX4、PKM2和GLUT1的表达水平变化;活性氧(ROS)荧光探针检测细胞内ROS阳性率变化;葡萄糖和乳酸检测试剂盒检测糖代谢原料葡萄糖(GLU)及产物乳酸(LD)水平;建立BALB/c裸鼠皮下肿瘤模型, 分析SKN对ATC在体内的抑制作用。结果与对照组相比, SKN处理后的CAL-62细胞内:NF-κB、GPX4、TXNRD1、GLUT1、PKM2蛋白表达水平下降(P=0.004,P=0.012,P=0.043,P=0.001,P=0.018);GPX4、GLUT1、PKM2的mRNA表达下降(P<0.001,P=0.029,P<0.001);ROS产生增多(P=0.041);铁死亡抑制剂Liproxstain-1(L-1)处理后细胞内一定比例死亡被逆转, L-1处理后细胞死亡比例无统计学意义, 细胞内发生铁死亡(P<0.001);GLU摄取量及LD生成量减少(P<0.001);SKN在体抑制ATC肿瘤生长(P=0.016)。结论 SKN促进ATC细胞内铁死亡, 抑制糖酵解作用及葡萄糖摄取, 抑制ATC细胞生长。
Objective To explore the role and molecular mechanism of Shikonin(SKN)in inhibiting the growth of anaplastic thyroid carcinoma(ATC)cells.Methods The effect of SKN on ferroptosis in ATC cell lines CAL-62 was detected by flow cytometry;the expression levels of NF-κB,ferroptosis-related genes glutathione peroxidase 4(GPX4)and selenoprotein thioredoxin reductase 1(TXNRD1),glucose metabolism-related genes pyruvate kinase isoform 2(PKM2)and glucose transporter protein 1(GLUT1)were detected by Western blotting;real-time fluorescence quantitative(qPCR)to detect changes in the expression levels of GPX4,PKM2 and GLUT1;reactive oxygen species(ROS)fluorescent probe to detect changes in intracellular ROS positivity;glucose and lactic acid assay kit to detect the levels of glucose,the raw material of glucose metabolism(GLU),and lactate(LD),the product of glucose metabolism;and establishment of a subcutaneous tumour model in BALB/c nude mice to analyse the inhibitory effect of SKN on ATC in vivo.Results Compared to the control group,after SKN treatment,the protein expression levels of NF-κB,GPX4,TXNRD1,GLUT1,and PKM2 in CAL-62 cells decreased(P=0.004,P=0.012,P=0.043,P=0.001,P=0.018);the mRNA expression of GPX4,GLUT1,and PKM2 also decreased(P<0.001,P=0.029,P<0.001).Additionally,ROS production increased(P=0.041).After treatment with the ferroptosis inhibitor Liproxstatin-1(L-1),the proportion of cell death was reversed to a certain extent,and there was no statistically significant difference in cell death proportion after L-1 treatment.Intracellular ferroptosis occurred(P<0.001),with reduced levels of glutamate(GLU)uptake and lipid peroxidation(LD)generation(P<0.001).SKN inhibited ATC tumor growth in vivo(P=0.016).Conclusion SKN promotes intracellular ferroptosis in ATC cells,inhibits glycolysis and glucose uptake,and suppresses ATC cell growth.
作者
杨晨
杨磊
李棣华
王艳
谭建
贾强
孟召伟
Yang Chen;Yang Lei;Li Dihua;Wang Yan;Tan Jian;Jia Qiang;Meng Zhaowei(Department of Nuclear Medicine,General Hospital of Tianjin Medical University,Tianjin 300052,China;Acute Abdominal Disease Related Organ Injury and ITCWM Repair,Tianjin Nankai Hospital,Tianjin 300102,China;College of Pharmaceutical Engineering of Traditional Chinese Medicine,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China)
出处
《中华内分泌代谢杂志》
CAS
CSCD
北大核心
2024年第5期420-426,共7页
Chinese Journal of Endocrinology and Metabolism
基金
国家自然科学基金(81971650)
天津市科学技术委员会基金(21JCYBJC01820)。
