米贝地尔增强3,3′二吲哚甲烷对肝癌细胞的抑制作用  

Mibefradil enhances inhibition of 3,3′-diindolylmethane in hepatocellular carcinoma cells

作　　者：姜袁越 徐伟 顾杰 叶洋[1] 徐新明[4] 孙俊[4] 陈健[4] 陆荣柱[1,5] JIANG Yuanyue;XU Wei;GU Jie;YE Yang;XU Xinming;SUN Jun;CHEN Jian;LU Rongzhu(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013;Department of Pathology,Kunshan Hospital of Traditional Chinese Medicine,Suzhou Jiangsu 215300;Institute of Life Sciences,Jiangsu University,Zhenjiang Jiangsu 212013;Department of General Surgery,Affiliated Kunshan Hospital of Jiangsu University,Suzhou Jiangsu 215300;Center for Experimental Research,Affiliated Kunshan Hospital of Jiangsu University,Suzhou Jiangsu 215300,China)

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]昆山市中医医院病理科,江苏苏州215300 [3]江苏大学生命科学研究院,江苏镇江212013 [4]江苏大学附属昆山医院普外科,江苏苏州215132 [5]江苏大学附属昆山医院临床实验研究中心,江苏苏州215300

出　　处：《江苏大学学报（医学版）》2021年第4期333-338,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81502800,31271272,31600952);中国博士后科学基金资助项目(2015M571694);江苏省博士后科研基金资助项目(1402169C)。

摘　　要：目的:探讨T型钙通道阻滞剂米贝地尔对3,3′二吲哚甲烷(3,3′-diindolylmethane,DIM)抗肝癌效应的影响及其潜在的分子机制。方法:选用肝癌SMMC-7721和HepG2细胞,分别设4组,对照组用含10%胎牛血清的高糖培养基培养24.5 h;米贝地尔组用5μmol/L米贝地尔处理24.5 h;DIM组用60μmol/L DIM处理24.5 h;米贝地尔+DIM组用5μmol/L米贝地尔预处理0.5 h,再与60μmol/L DIM共同处理24 h;采用倒置显微镜观察细胞的形态学变化;采用CCK-8法检测各组细胞增殖活力;Hoechst 33342染色观察细胞凋亡水平,蛋白免疫印迹法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、裂解的半胱氨酸蛋白酶3(cleaved-caspase 3)以及磷酸化p38丝裂原活化蛋白激酶(phosphorylation of p38 mitogen activated protein kinase,p-p38 MAPK)表达水平;以Fluo-3/AM为探针,观察肝癌细胞胞质游离钙(cytosolic free calcium,[Ca^(2+)]i)水平。结果:与对照组相比,DIM组细胞增殖活力下降,PCNA表达明显降低,凋亡水平升高,cleaved-caspase 3、p-p38 MAPK表达水平明显升高(P均<0.05);部分细胞形态消失,[Ca^(2+)]i荧光增强。与DIM组相比,米贝地尔+DIM组细胞增殖活力明显降低,PCNA表达明显下降,凋亡水平进一步升高,且cleaved-caspase 3、p-p38 MAPK表达水平进一步升高(P均<0.05);多数细胞形态消失,[Ca^(2+)]i荧光增强更明显。结论:T型钙通道阻滞剂米贝地尔可增强DIM抗肝癌作用,可能与[Ca^(2+)]i水平增加以及p-p38 MAPK表达升高有关。Objective:To investigate the effect of T-type calcium channel blocker mibefradil on anti-cancer effects of 3,3′-diindolylmethane(DIM)and its potential molecular mechanism in human hepatocellular carcinoma cells.Methods:SMMC-7721 and HepG2 hepatocellular carcinoma cells were cultured in four groups,control group,cultured in high glucose medium containing 10%fetal bovine serum for 24.5 h;mibefradil group,cells were treated with 5μmol/L mibefradil for 24.5 h;DIM group,cells were treated with 60μmol/L DIM for 24.5 h;mibefradil+DIM group,cells were pretreated with 5μmol/L mibefradil for 0.5 h,then co-treated with 5μmol/L mibefradil and 60μmol/L DIM in the following 24 h.The morphological changes were observed by the inverted microscope;the cell viability was detected by CCK-8 assays;the level of apoptosis was detected by Hoechst 33342 staining;the expression of PCNA,cleaved-caspase 3 and p-p38 MAPK were evaluated by Western blotting;Fluo-3/AM was used as the probe to assess the level of cytosolic free calcium[Ca^(2+)]i.Results:Compared with the control group,the treatment with DIM decreased the cell viability and the expression of PCNA,whereas enhanced the expression of cleaved-caspase 3 and p-p38 MAPK(all P<0.05);the cell morphology was destroyed and the[Ca^(2+)]i fluorescence was enhanced.Compared with the DIM group,the cell viability and the expression of PCNA was lower in mibefradil+DIM group;the level of apoptosis,the expression of cleaved-caspase 3 and p-p38 MAPK was further increased(all P<0.05);cell morphology was further destroyed,the level of[Ca^(2+)]i was muh higher.Conclusion:T-type calcium channel blocker mibefradil could enhance the anti-cancer effects of DIM through the increase of[Ca^(2+)]i and p-p38 MAPK.

关 键 词：T型钙通道阻滞剂 3 3′二吲哚甲烷 肝癌细胞 胞质游离钙 P38丝裂原活化蛋白激酶 米贝地尔 

分 类 号：R735.7[医药卫生—肿瘤]