洋地黄毒苷抑制PI3K/AKT信号通路对下咽癌Fadu细胞增殖和凋亡影响  

Effect of Digitoxin on the proliferation and apoptosis of hypopharyngeal carcinoma Fadu cells through PI3K/AKT signaling pathway

作　　者：陈帅[1] 刘恒[1] CHEN Shuai;LIU Heng(NHC Key Laboratory of Otorhinolaryngology,Department of Otorhinolaryngology,Qilu Hospital of Shandong University,Jinan 250012,China)

机构地区：[1]山东大学齐鲁医院国家卫建委耳鼻喉科学重点实验室,山东大学齐鲁医院耳鼻喉科,山东济南250012

出　　处：《社区医学杂志》2021年第9期553-558,共6页Journal Of Community Medicine

摘　　要：目的探讨强心苷药物洋地黄毒苷对下咽癌Fadu细胞增殖和凋亡的影响,并初步探讨其可能的作用机制。方法 0、50、100、200和400nmol/L的洋地黄毒苷不同时间点处理Fadu细胞,细胞计数试剂(CCK-8)和三磷酸腺苷(ATP)发光法检测Fadu细胞增殖,Annexin Ⅴ-FITC/PI双标流式细胞术和Caspase3/7活细胞荧光实时法检测细胞凋亡,蛋白质印迹法检测PI3K和AKT总蛋白和磷酸化蛋白水平。结果细胞增殖检测结果显示,洋地黄毒苷剂量和时间依赖性抑制FaDu细胞增殖,48h后,CCK-8检测值分别为0.73±0.02、0.63±0.02、0.43±0.02和0.23±0.02,低于对照组的1.27±0.05,F浓度=17 810.000,P<0.001;F时间=230.900,P<0.001;F时间×浓度=292.100,P<0.001,IC50值为102nmol/L。而ATP检测值为360 175.00±15 432.88、277 193.00±20 311.25、212 869.66±9 955.65和164 726.66±12 536.46,低于对照组的682 460.66±37 594.12,F浓度=345.600,P=0.003;F时间=120.700,P<0.001;F时间×浓度=95.000,P<0.001。细胞凋亡检测结果显示,随着洋地黄毒苷浓度增加,FaDu细胞的凋亡率增加到(20.42±1.54)%和(34.58±2.16)%,高于对照组的(11.94±1.39)%,F浓度=130.100,P<0.001;而凋亡相关蛋白Caspase3/7的活性增加到33 986.00±2 513.50和43 786.33±1 736.32,高于对照组的22 652.66±2 162.53;F浓度=71.860,P<0.001。蛋白质印迹法分析结果显示,洋地黄毒苷能剂量性的降低p-PI3K蛋白到0.51±0.04和0.18±0.002,低于对照组的1.00±0.07,F浓度=276.400,P<0.001;而p-AKT蛋白降低到0.46±0.03和0.13±0.004,低于对照组1.00±0.05,F浓度=483.500,P<0.001。结论洋地黄毒苷可能通过抑制PI3K/AKT信号通路诱导下咽癌细胞的凋亡,抑制细胞增殖。Objective To investigate the effect of the cardiac glycoside drug,digoxigenin,on the proliferation and apoptosis of hypopharyngeal carcinoma Fadu cells,and to explore preliminarily the possible mechanism.Methods Fadu cells were treated at 0、50、100、200 and 400 nmol/L concentrations of digitoxin for different times,cell proliferation was measured by CCK8 assay and adenosine triphosphate(ATP)luminescence assay,and the percentage of apoptotic cells was analyzed with AnnexinⅤ-FITC/PI double-staining flow cytometry and Caspase3/7 live cell fluorescence assay.Total protein and phosphorylated protein of PI3 Kand AKT was detected by western blotting.Results The results of cells proliferation showed that digitoxin inhibited the proliferation of FaDu cells in a dose-and time-dependent manner.After 48 hours,the values of CCK-8 detection were 0.73±0.02,0.63±0.02,0.43±0.02 and 0.23±0.02,which were lower than the control group 1.27±0.05(Fconcentration=17 810.000,P<0.001,Ftime=230.900,P<0.001,Ftime×concentration=292.100,P<0.001),and the IC50 value was 102 nmol/L.The ATP detection values were 360 175.00±15 432.88,277 193.00±20 311.25,212 869.66±9 955.65 and 164 726.66±12 536.46,which were lower than the control group 682 460.66±37 594.12(Fconcentration=345.600,P=0.003,Ftime=120.700,P<0.001,Ftime×concentration=95.000,P<0.001).The results of cells apoptosis showed that with increasing concentration of digitoxin the percentage of apoptotic FaDu cells increased to(20.42±1.54)% and(34.58±2.16)%,which were higher than the control group(11.94±1.39)%(Fconcentration=130.100,P<0.001).The activity of the apoptosis-related protein Caspase3/7 was increased to 33 986.00±2 513.50 and43 786.33±1 736.32,which were higher than the control group 22 652.66±2 162.53(Fconcentration=71.860,P<0.001).The western blot result showed that digitoxin can downregulated the phosphorylation level of PI3 Kto 0.51±0.04 and0.18±0.002 in a dose manner,which were lower than the control group 1.00±0.07(Fconcentration276.400,P<0.001),and p-

关 键 词：PI3K/AKT 细胞凋亡 细胞增殖 下咽癌 洋地黄毒苷 

分 类 号：R739.63[医药卫生—肿瘤]