摘要/Abstract

摘要： 目的·研究钠钙交换体（Na⁺/Ca²⁺ 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，IC₅₀）。钙成像技术检测NCX阻滞剂处理后U87细胞内Ca²⁺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，IC₅₀值分别为2.06、2.19 和1.82 μmol/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Ca²⁺ 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。
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
Abstract:
Objective·To test the inhibitory effect of the Na⁺/Ca²⁺ 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 (IC₅₀) of the drug was obtained. Calcium imaging was used to detect the changes of Ca²⁺ 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, U251 and SF188) for 48 h caused a dose-dependent growth inhibition with IC₅₀ values of 2.06, 2.19 and 1.82 μmol/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 Ca²⁺, 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 glioblastoma cells may be related to blocking the forward transport mode of NCX. The plasma membrane NCX is a potential new target for the treatment of human glioblastoma.

Key words: glioblastoma, Na⁺/Ca²⁺ exchanger, blocker, CB-DMB, apoptosis, temozolomide (TMZ)


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