乙酰紫草素诱导急性髓系白血病HL-60细胞发生铁死亡的作用及分子机制  

Effect and molecular mechanism of acetylshikonin-induced ferroptosis in acute myelogenous leukemia HL-60 cells

作　　者：李子辉 周智辉 王蓉 李媛媛[2] 李宏[1,2,3] LI Zihui;ZHOU Zhihui;WANG Rong;LI Yuanyuan;LI Hong(Shaanxi University of Chinese Medicine,Shaanxi Xianyang 712046,China;The Second Affiliated Hospital of Shaanxi University of Chinese Medicine,Shaanxi Xianyang 712000,China;Pharmaceutical Factory,Shaanxi University of Chinese Medicine,Shaanxi Xianyang 712000,China.)

机构地区：[1]陕西中医药大学,陕西咸阳712046 [2]陕西中医药大学第二附属医院,陕西咸阳712000 [3]陕西中医药大学制药厂,陕西咸阳712000

出　　处：《现代肿瘤医学》2023年第14期2581-2588,共8页Journal of Modern Oncology

基　　金：陕西省自然科学基础研究计划(编号:2019JM-224);陕西省卫生健康科研项目(编号:2018D087);陕西省卫生科研项目(编号:2016D030);陕西中医药大学学科创新团队项目(编号:2019-YS03)。

摘　　要：目的:探究乙酰紫草素诱导急性髓系白血病HL-60细胞发生铁死亡的作用及分子机制。方法:采用1μg/mL、2μg/mL、4μg/mL和8μg/mL的乙酰紫草素干预HL-60细胞24 h和48 h,CCK-8实验检测乙酰紫草素对HL-60细胞增殖活力的影响;Western blot检测铁死亡相关蛋白[转录调节因子p53、胱氨酸/谷氨酸反向转运体(XCT)、谷胱甘肽过氧化物酶4(GPX4)、膜蛋白转铁蛋白受体1(TFR1/CD71)、铁蛋白重链1(FTH1)]的表达水平;采用GSH/GSSG检测试剂盒检测不同浓度乙酰紫草素干预HL-60细胞24 h后细胞内GSH、GSSG的含量变化;采用4μg/mL的乙酰紫草素和0.1μmol/L细胞铁死亡抑制剂(Ferrostatin-1,Fer-1)共同干预HL-60细胞24 h,Western blot检测铁死亡相关蛋白p53、XCT、GPX4、CD71、FTH1的表达水平。结果:与对照组和DMSO组相比,不同浓度的乙酰紫草素对HL-60细胞的增殖均具有较强的抑制作用;乙酰紫草素显著升高p53和CD71的表达水平,显著降低XCT、GPX4和FTH1的表达水平,其中4μg/mL乙酰紫草素干预HL-60细胞24 h,各蛋白表达水平变化最明显;乙酰紫草素显著减少GSH的含量(P<0.0001),显著提高GSSG/GSH的比值(P<0.0001);Fer-1将显著升高的p53和CD71以及显著降低的XCT、GPX4和FTH1的表达水平逆转。结论:乙酰紫草素一方面可能通过上调p53的表达,抑制XCT的表达,减少GSH含量,进而抑制GPX4的表达,促进HL-60细胞发生铁死亡;另一方面通过上调CD71(TFR1)的表达,下调FTH1的表达,从而影响HL-60细胞内铁稳态促进铁死亡的发生。Objective:To explore the effect and molecular mechanism of acetylshikonin-induced ferroptosis in acute myelogenous leukemia HL-60 cells.Methods:HL-60 cells were intervened with 1μg/mL,2μg/mL,4μg/mL and 8μg/mL of acetylshikonin for 24 h and 48 h,and the effect of acetylshikonin on the proliferation of HL-60 cells was detected by CCK-8 assay.Western blot detected the expression levels of ferroptosis-related proteins,such as,transcriptional regulator p53,cystine/glutamate antiporter(XCT),glutathione peroxidase 4(GPX4),membrane protein transferrin receptor 1(TFR1/CD71)and ferritin heavy chain 1(FTH1).GSH/GSSG detection kit was used to detect the changes of intracellular GSH and GSSG contents in HL-60 cells after 24 h intervention with different concentrations of acetylshikonin.HL-60 cells were co-treated with 4μg/mL acetylshikonin and 0.1μmol/L Ferrostatin-1(Fer-1)for 24 h,and the expression levels of ferroptosis-related proteins,such as,p53,XCT,GPX4,CD71 and FTH1 were detected by Western blot.Results:Compared with the control group and the DMSO group,different concentrations of acetylshikonin had strong inhibitory effects on the proliferation of HL-60 cells.Acetylshikonin significantly increased the expression levels of p53 and CD71,significantly decreased the expression levels of XCT,GPX4 and FTH1,among which,4μg/mL acetylshikonin intervened HL-60 cells for 24 h,and the expression level of each protein changed most obviously.Acetylshikonin significantly reduced the content of GSH(P<0.0001)and significantly increased the ratio of GSSG/GSH(P<0.0001).Fer-1 reversed markedly elevated expression levels of p53 and CD71 and markedly reduced expression levels of XCT,GPX4 and FTH1.Conclusion:On the one hand,acetylshikonin may promote ferroptosis in HL-60 cells by up-regulating the expression of p53,inhibiting the expression of XCT,reducing the content of GSH,then inhibiting the expression of GPX4.On the other hand,acetylshikonin may affect iron homeostasis in HL-60 cells and promote ferroptosis by up-regulating the e

关 键 词：乙酰紫草素 HL-60细胞 细胞铁死亡 

分 类 号：R733.71[医药卫生—肿瘤]