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    ADAM10抑制剂GI254023X对CCRF-CEM和Molt4细胞增殖与凋亡的作用及机制研究

    Effect of ADAM10 inhibitor GI254023X on proliferation and apoptosis of acute T-lymphoblastic leukemia CCRF-CEM and Molt4 cells and its possible mechanisms

    • 摘要: 目的 本研究旨在探讨解聚素金属基质蛋白酶10(ADAM10)抑制剂GI254023X对急性T淋巴细胞白血病CCRF-CEM及Molt4细胞株增殖、凋亡的影响及其作用机制。方法 选择不同浓度的GI254023X处理CCRF-CEM及Molt4细胞,CCK-8法检测并绘制增殖抑制曲线,Annexin V/7-AAD双标记法分析细胞凋亡情况,流式细胞术检测细胞周期的变化,Western blot检测切割后的Notch1蛋白(Cleaved Notch1),定量PCR检测抑凋亡基因Bcl-2c-MycMcl-1,促凋亡基因BAD、BAK及Notch1靶基因Hes-1的转录变化。结果 GI254023X能明显抑制CCRF-CEM及Molt4细胞增殖,且抑制效应呈一定的时间和剂量依赖性(CCRF-CEM 细胞24、48 h的相关系数r分别为0.981、0.962, Molt4细胞24、48 h的相关系数r分别为0.992、0.957),CCRF-CEM细胞24、48 h的半数抑制浓度(IC50)分别为(29.40±3.28)μmol/L和(11.14±2.21)μmol/L;Molt4细胞24、48 h的IC50分别为(19.04±1.58)μmol/L和(11.25±2.31)μmol/L;GI254023X能够诱导CCRF-CEM及Molt4细胞凋亡,使细胞阻滞于G0/G1期;Western blot检测结果显示,Cleaved Notch1在GI254023X作用后水平下降;GI254023X能够使抑凋亡基因Bcl-2c-Myc、Mcl-1Hes-1表达减低,促凋亡基因BAD、BAK表达升高。结论 GI254023X能抑制CCRF-CEM及Molt4细胞增殖并诱导其凋亡,其机制可能与抑制Notch1活化有关。

       

      Abstract: Objective To investigate the effect of GI254023X, an ADAM10 (a desintegrin and metalloproteinase domain containing protein 10)inhibitor, on the proliferation and apoptosis of CCRF-CEM and Molt4 cell lines and its mechanisms. Methods CCRF-CEM and Molt4 cells were treated with different concentrations of GI254023X. Proliferation-inhibition curve was assayed and plotted by CCK-8 method. Cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining, and cell cycle changes were analyzed by flow cytometry. For mechanism exploration, the cleavage of Notch1 protein was determined by Western blot. The transcripts of anti-apoptotic genes Bcl-2, c-Myc, Mcl-1 , pro-apoptosis genes BAD, BAK and Notch1 target gene Hes-1 were detected by real-time PCR. Results GI254023X inhibited the proliferation of CCRF-CEM and Molt4 cells in a time-and dose-depended manner (the rvalues of CCRF-CEM cells for 24 and 48 h were 0.981 and 0.962, respectively, while r values of Molt4 cells for 24 and 48 h were 0.992 and 0.957, respectively). The half maximal inhibitory concentration (IC50) values of CCRF-CEM cells for 24 and 48 h were (29.40±3.28) μmol/L and (11.14±2.21) μmol/L, respectively, while IC50 values of Molt4 cells for 24 and 48 h were (19.04±1.58) μmol/L and (11.25±2.31) μmol/L, respectively. Compared with the control group,the apoptosis rate of cells increased with the increment of concentration of GI254023X. Cell cycles were arrested at G0/G1 phase. Compared with DMSO in the control group, the expression of cleaved Notch1 was down-regulated after the treatment with GI254023X.The mRNA levels of anti-apoptotic genes Bcl-2 , c-Myc, Mcl-1 and Notch1 target gene Hes-1 mRNA transcripts in CCRF-CEM and Molt4 cells were reduced in GI254023X treated group, while the mRNA levels of pro-apoptosis genes BAD, BAK increased. Conclusions GI254023X can inhibit the proliferation and induce apoptosis of CCRF-CEM and Molt4 cells.Its mechanism may be related to the inhibition of Notch1 pathway.

       

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