伊曲康唑促进AMPK磷酸化诱发结直肠癌细胞铁死亡的抗癌机制研究  

Itraconazole suppresses the proliferation of colorectal cancer HCT116 cells via promoting AMPK mediated ferroptosis

作　　者：李慧霞[1] 尤伟波[2] 陈丽[1] 王建平[1] 邵初晓[1] LI Huixia;YOU Weibo;CHEN Li;WANG Jianping;SHAO Chuxiao(Department of Anorectal Surgery,Lishui Hospital of TCM(the Fifth Affiliated Hospital of Wenzhou Medical University),Lishui 323000,China)

机构地区：[1]丽水市中心医院(温州医科大学附属第五医院)肛肠外科,323000 [2]丽水市中心医院(温州医科大学附属第五医院)检验科,323000

出　　处：《浙江医学》2021年第24期2619-2622,2626,I0014,共6页Zhejiang Medical Journal

基　　金：浙江省医药卫生科技计划项目(2020379262)。

摘　　要：目的探讨伊曲康唑(Itra)促进腺苷酸蛋白激酶(AMPK)磷酸化诱发结直肠癌(CRC)细胞铁死亡的抗癌机制。方法体外培养结肠癌HCT116细胞,并将细胞分为对照组、Itra组、Itra+AMPK抑制剂组(抑制剂组)和Itra+AMPK siRNA干扰组(干扰组);采用MTT法检测细胞存活率,活死细胞染色法检测死亡细胞比例,Western blot法检测AMPK、磷酸化AMPK(p-AMPK)、胱甘肽过氧化物酶4(GPX4)、xCT蛋白表达水平,流式细胞术检测细胞内活性氧(ROS)含量,Hoechst染色法检测细胞凋亡率。结果与0μmol/L组比较,0.5、1.0、2.5、5.0、10.0、25.0、50.0、100.0μmol/L的Itra处理48 h后,HCT116细胞存活率均明显下降(均P<0.05),IC_(50)为(4.86±1.23)μmol/L。活死细胞染色显示,2.5、5.0、10.0μmol/L的Itra处理48 h后,死亡细胞比例较0μmol/L组均明显增加(均P<0.05)。与0μmol/L组比较,2.5、5.0、10.0μmol/L的Itra处理48 h后,HCT116细胞p-AMPK蛋白表达水平均明显增加(均P<0.05);以5.0μmol/L为Itra的终浓度处理HCT116细胞12、24、48 h后,p-AMPK蛋白表达水平亦明显增加(均P<0.05)。与对照组比较,5.0μmol/L的Itra处理48 h后HCT116细胞内ROS含量及细胞凋亡率均明显增加(均P<0.05),GPX4、xCT蛋白表达水平均明显下降(均P<0.05);与Itra组比较,抑制剂组、干扰组细胞内ROS含量及细胞凋亡率均明显下降(均P<0.05),GPX4、xCT蛋白表达水平均明显增加(均P<0.05)。结论体外研究证实Itra通过激活AMPK信号通路来增加CRC细胞内ROS含量,进而抑制GPX4、xCT蛋白表达,促使铁死亡的发生,最终抑制CRC细胞的增殖。Objective To investigate the effect of itraconazole(Itra)on ferroptosis of colorectal cancer cells and its mechanism.Methods Colorectal cancer HCT116 cells were divided into control group,Itra group,Itra+AMPK inhibitor group(inhibitor group)and Itra+AMPK siRNA interference group(interference group).Cell proliferation was detected by MTT,cell survival was detected by living and dead cell staining kit.The expression of AMPK,phosphorylated AMPK(p-AMPK),glutathione peroxidase 4(GPX4)and xCT were detected by Western blot.Intracellular ROS were analyzed using flow cytometry,apoptotic cells were detected by Hoechst staining.Results Compared with control group,the survival rate of HCT116 cells decreased significantly in Itra group treated with dosage of 0.5,1.0,2.5,5.0,10.0,25.0,50.0 and 100.0μmol/L Itra for 48 h(all P<0.05)and the half-inhibitory concentration(IC_(50))was(4.86±1.23)μmol/L.HCT116 cells treated with 2.5,5.0,10.0μmol/L Itra resulted in increased expression of p-AMPK and death cells rate in a dosage dependent manner.The treatment of HCT116 cells with 5.0μmol/L Itra for 12,24,48 h led to increased expression of p-AMPK in a time dependent manner(all P<0.05).Compared with the control group,the cell death rate,ROS content increased while the expression of GPX4 and xCT decreased in Itra group(both P<0.05).Compared with Itra group,the cell death rate,ROS content decreased but the expression of GPX4 and xCT increased in inhibitor group and interference group(all P<0.05).Conclusion Itra can suppress the proliferation of colorectal HCT116 cells via promoting AMPK mediated ferroptosis.

关 键 词：伊曲康唑 铁死亡 腺苷酸蛋白激酶 结直肠癌 

分 类 号：R735.34[医药卫生—肿瘤]