SF2523对人源脑胶质瘤干细胞TS576增殖的抑制作用及其机制

目的：探讨PI3K和BRD4双重抑制剂SF2523对人源脑胶质瘤干细胞TS576增殖的抑制作用，并初步阐明其作用机制。方法：培养人源脑胶质瘤干细胞TS576，将其分为对照组和0.25、0.50、1.00、2.00 μmol·L^-1 SF2523组。采用CCK-8法检测各组TS576细胞存活率；将TS576细胞分为对照组和2 μmol·L^-1 SF2523组，利用细胞生长计数法检测各组TS576细胞数；将TS576细胞分为对照组和1及2 μmol·L^-1 SF2523组，采用流式细胞术检测各组不同细胞周期TS576细胞百分比；将TS576细胞分为对照组和1及2 μmol·L^-1 SF2523组，利用Annexin Ⅴ/PI染色法检测各组TS576细胞凋亡率；将TS576细胞分为对照组和1及2 μmol·L^-1 SF2523组，采用Western blotting法检测各组TS576细胞中B淋巴细胞瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）和cyclinD1蛋白表达水平。结果：CCK-8检测，作用24、48和72 h时，与对照组比较，0.25、0.50、1.00和2.00 μmol·L^-1 SF2523组TS576细胞存活率均明显降低（P<0.01）。细胞生长计数法检测，与对照组比较，2 μmol·L^-1 SF2523组细胞数明显减少（P<0.05或P<0.01）。流式细胞术检测，作用72 h时，与对照组比较，1和2 μmol·L^-1 SF2523组G₁期TS576细胞百分比升高（P<0.05），S期TS576细胞百分比降低（P<0.05）。Annexin Ⅴ/PI染色检测，作用72 h时，与对照组比较，1和2 μmol·L^-1 SF2523组细胞凋亡率均明显升高（P<0.01）。Western blotting法检测，作用72 h时，与对照组比较，1和2 μmol·L^-1SF2523组TS576细胞中Bax蛋白表达水平升高（P<0.01），Bcl-2和cyclinD1蛋白表达水平降低（P<0.05或P<0.01），Bax/Bcl-2比值升高（P<0.01）。结论：SF2523通过下调cyclinD1表达引起TS576细胞G₁期阻滞，通过上调Bax表达、下调Bcl-2表达促进TS576细胞凋亡，从而抑制TS576细胞增殖。

Inhibitory effect of SF2523 on proliferation of human glioma stem cells TS576 and its mechanism

Objective: To investigate the inhibitory effect of SF2523, a dual inhibitor of BRD4 and PI3K, on the proliferation of human glioma stem cells TS576, and to preliminarily elucidate its mechanism. Methods: The human glioma stem cells TS576 were cultured and divided into control group and 0.25, 0.50,1.00 2.00 μmol·L^-1 SF2523 groups,and CCK-8 method was used to detect the survival rates of TS576 cells.The TS576 cells were divided into control group and 2 μmol·L^-1 SF2523 group,and the number of TS576 cells in various groups was detected by cell growth counting method.The TS576 cells were divided into control group, 1 and 2 μmol·L^-1 SF2523 groups,and the percentages of TS576 cells at different cell cycles in various groups were examined by flow cytometry.The TS576 cells were divided into control group, 1 and 2 μmol·L^-1 SF2523 groups,and the apoptotic rates of TS576 cells in various groups were detected by Annexin Ⅴ/PI staining. The TS576 cells were divided into control group, 1 and 2 μmol·L^-1 SF2523 groups, and the expression levels of cyclinD1, B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein (Bax)proteins in the TS576 cells in various groups were detected by Western blotting method. Results: Compared with control group, the proliferation rates of TS576 cells in 0.25, 0.50,1.00 and 2.00 μmol·L^-1 SF2523 groups at 24, 48 and 72 h after treatment were significantly decreased (P<0.01).Compared with control group, the number of TS576 cells in 2 μmol·L^-1 SF2523 group was decreased significantly (P<0.05 or P<0.01). Compared with control group, the percentages of TS576 cells at G₁ phase in 1 and 2 μmol·L^-1 SF2523 groups were increased (P<0.05), the percentage of TS576 cells at S phase was decreased (P<0.05).The results of Annexin Ⅴ/PI staining showed that the apoptotic rates of TS576 cells in 1 and 2 μmol·L^-1 SF2523 groups were significantly increased at 72 h after treatment compared with control group (P<0.01). The results of Western blotting method showed that the expression levels of Bax protein in the TS576 cells in 1 and 2 μmol·L^-1 SF2523 groups were significantly increased (P<0.01), the expression levels of Bcl-2 and cyclinD1 were significantly decreased (P<0.05 or P<0.01), and the ratio of Bax/bcl-2 was also significantly increased (P<0.01). Conclusion: SF2523 can induce the G₁ phase arrest of TS576 cells by down-regulating the expression of cyclinD1 and promote the apoptosis of TS576 cells by up-regulating the expression of Bax and down-regulating the expression of Bcl-2, and then inhibit the proliferation of TS576 cells.