米贝地尔增强3，3′二吲哚甲烷对肝癌细胞的抑制作用

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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5 μmol/L米贝地尔处理24.5 h；DIM组用60 μmol/L DIM处理24.5 h；米贝地尔+DIM组用5 μmol/L米贝地尔预处理0.5 h，再与60 μmol/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，［Ca2+］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，［Ca2+］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，［Ca2+］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［Ca2+］i水平增加以及pp38 MAPK表达升高有关。

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	姜袁越1
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	陈健4
	陆荣柱1
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关键词 ： T型钙通道阻滞剂, 3, 3&prime, 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

Abstract：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 5 μmol/L mibefradil for 24.5 h; DIM group, cells were treated with 60 μmol/L DIM for 24.5 h; mibefradil+DIM group, cells were pretreated with 5 μmol/L mibefradil for 0.5 h, then cotreated with 5 μmol/L mibefradil and 60 μmol/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［Ca2+］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［Ca2+］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 ［Ca2+］i was muh higher. Conclusion: Ttype calcium channel blocker mibefradil could enhance the anticancer effects of DIM through the increase of［Ca2+］i and pp38 MAPK.

收稿日期: 2020-02-20

基金资助: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）

通讯作者: 陆荣柱（通讯作者），教授，博士生导师，E-mail: lurz@ujs.edu.cn

作者简介: 姜袁越（1993—），女，硕士研究生

引用本文:

姜袁越1,2，徐伟1，顾杰3，叶洋1，徐新明4，孙俊4，陈健4，陆荣柱1,5. 米贝地尔增强3，3′二吲哚甲烷对肝癌细胞的抑制作用[J]. 江苏大学学报:医学版, 2021, 31(04): 333-338. JIANG Yuanyue1,2, XU Wei1, GU Jie3, YE Yang1, XU Xinming4, SUN Jun4, CHEN Jian4, LU Rongzhu1,5.

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

. Journal of Jiangsu University(Medicine Edition), 2021, 31(04): 333-338.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2021/V31/I04/333

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