钠钙交换体阻滞剂CB-DMB对人胶质母细胞瘤细胞生长的抑制作用  

Suppressing effect of the Na^(+)/Ca^(2+) exchanger blocker CB-DMB on the growth of human glioblastoma cells

作　　者：刘景景 胡慧洁 刘子楷 宋明柯 LIU Jing-jing;HU Hui-jie;LIU Zi-kai;SONG Ming-ke(Shanghai Jiao Tong University College of Basic Medical Sciences,Shanghai 200025,China)

机构地区：[1]上海交通大学基础医学院药理学与化学生物学系,上海200025

出　　处：《上海交通大学学报（医学版）》2021年第6期710-716,共7页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81873807,91949116,81671375);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20181201)。

摘　　要：目的·研究钠钙交换体(Na^(+)/Ca^(2+)exchanger,NCX)阻滞剂对人胶质母细胞瘤生长的抑制作用。方法·体外培养人胶质母细胞瘤细胞(U87、U251、SF188)和星形胶质细胞,用NCX阻滞剂SN-6、YM244769、SEA0400、5-(N-4-氯苄基)-N-(2’,4’-二甲基)氨苯蝶啶(CB-DMB)以及化学治疗药物替莫唑胺(temozolomide,TMZ)处理细胞。SN-6、YM244769和SEA0400是NCX反向模式的选择性阻滞剂;CB-DMB是NCX双向模式的选择性阻滞剂,但优先阻滞NCX的正向模式;TMZ作为参考。CCK-8实验测定细胞生长活性,并分析药物对胶质母细胞瘤细胞的半数抑制浓度(half maximal inhibitory concentration,IC_(50))。钙成像技术检测NCX阻滞剂处理后U87细胞内Ca^(2+)信号的变化,Western blotting检测促分裂原活化的蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路蛋白的表达,流式细胞术检测NCX阻滞剂对细胞凋亡的影响。结果·CCK-8实验结果表明,与胶质母细胞瘤细胞(U87、U251、SF188)共孵育48 h后,NCX双向阻滞剂CB-DMB呈剂量依赖性地抑制肿瘤细胞的生长活性,IC_(50)值分别为2.06、2.19和1.82μmol/L;而NCX反向阻滞剂SN-6、YM244769和SEA0400对胶质母细胞瘤细胞的生长活性均无显著影响。CB-DMB对星形胶质细胞生长活性的影响较小。钙成像和Western blotting结果表明,CB-DMB通过阻滞NCX的正向模式导致U87细胞内Ca^(2+)增加,引起胞内钙超载,进而诱导U87细胞凋亡,并激活MAPK信号通路。流式细胞术结果表明,与TMZ相比,CB-DMB能够更快地引起U87细胞凋亡(P=0.002);CB-DMB与TMZ联合用药,增强了TMZ对肿瘤细胞生长活性的抑制作用。结论·CB-DMB对人胶质母细胞瘤的抑制作用可能与阻滞NCX的正向转运模式有关。细胞膜NCX可能成为胶质母细胞瘤治疗的潜在新靶点。Objective·To test the inhibitory effect of the Na^(+)/Ca^(2+) exchanger(NCX)blockers on the growth of human glioblastoma cells.Methods·Human glioblastoma cell lines(U87,U251 and SF188)and human astrocytes were cultured in vitro.The cells were treated with NCX blockers SN-6,YM244769,SEA0400,CB-DMB and the chemotherapeutic agent temozolomide(TMZ).SN-6,YM244769 and SEA0400 were selective inhibitors for the reverse operation of NCX;while CB-DMB was NCX bidirectional blocker,but preferentially blocked the forward mode of NCX.The TMZ was used as a reference drug.Cell counting kit-8(CCK-8)assay was used to quantify and assess the cell viability,and half maximal inhibitory concentration(IC_(50))of the drug was obtained.Calcium imaging was used to detect the changes of Ca^(2+) signal in U87 cells treated with NCX inhibitors,and Western blotting was used to detect the expression of mitogen-activated protein kinase(MAPK)signaling pathway proteins.Cellular apoptosis was evaluated by flow cytometry assay.Results·CCK-8 results showed that direct application of the NCX bidirectional blocker CB-DMB to glioblastoma cell lines(U87,U251and SF188)for 48 h caused a dose-dependent growth inhibition with IC_(50) values of 2.06,2.19 and 1.82μmol/L,respectively.In contrast,NCX reverse blockers SN-6,YM244769 and SEA0400 had no significant effect on the growth activity of glioblastoma cells.CB-DMB had little effect on the growth activity of human astrocytes.Calcium imaging and Western blotting results confirmed that CB-DMB blocked the forward transport mode of NCX to elevate intracellular Ca^(2+),causing intracellular calcium overload and then inducing apoptosis of U87 cells and activating MAPK signaling pathway.Flow cytometry assay results showed that the rate of apoptosis induced by CB-DMB in glioblastoma cells was much faster than that induced by TMZ(P=0.002).The combination of CB-DMB and TMZ enhanced the inhibitory effect of TMZ on the growth of tumor cells.Conclusion·The inhibitory effect of CB-DMB on the growth of human glioblas

关 键 词：胶质母细胞瘤 钠钙交换体 阻滞剂 5-(N-4-氯苄基)-N-(2’ 4’-二甲基)氨苯蝶啶(CB-DMB) 细胞凋亡 替莫唑胺 

分 类 号：R962[医药卫生—药理学]