CDK抑制剂BMS-265246体外诱导肝癌细胞周期阻滞和细胞凋亡

CDK inhibitor BMS-265246 induces cell cycle arrest and apoptosis of liver cancer cells in vitro

目的:筛选肝癌细胞Hep-3B和Hep-G2敏感的细胞周期蛋白质依赖激酶(cyclin-dependent kinase,CDK)抑制剂,并检测CDK抑制剂对肝癌细胞增殖和凋亡的影响。方法:采用CCK-8法检测10种CDK抑制剂对2株肝癌细胞Hep-3B和Hep-G2的增殖抑制率,选取抑制率较高的一种作为目标抑制剂,计算其对2株细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)。采用FCM法检测目标抑制剂处理后,肝癌Hep-3B和Hep-G2细胞周期分布及凋亡率的变化;进一步采用蛋白质印迹法检测细胞增殖及凋亡相关蛋白的表达水平变化。结果:6号抑制剂BMS-265246(5μmol/L作用48 h)对肝癌细胞Hep-3B和Hep-G2增殖的抑制率可达到80%,该药物对2株细胞的IC50值分别为2.84和1.73μmol/L。5μmol/L BMS-265246处理肝癌Hep-3B和Hep-G2细胞24 h后,G1期细胞所占百分率明显升高(P值均<0.05),细胞中磷酸化Rb蛋白的水平明显降低(P值均<0.01),转录因子E2F的靶蛋白c-Myc表达明显下调(P值均<0.01)。另外,5μmol/L BMS-265246对2株肝癌细胞均有明显的凋亡诱导作用,与未处理对照组相比,24 h和48 h凋亡率均明显升高(P值均<0.05);药物处理组细胞中抗凋亡蛋白Bcl-2和Bcl-xL的表达明显下调(P值均<0.05),而促凋亡蛋白Bax和Bak的表达明显上调(P值均<0.05),同时可检测出caspase-3及其底物聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)的活化片段。结论:CDK抑制剂BMS-265246在体外可诱导肝癌Hep-3B及Hep-G2细胞凋亡,并抑制细胞增殖;其作用机制可能与Bcl-2家族抗/促凋亡失衡、caspase通路激活,以及Rb去磷酸化和E2F活性受抑制有关。

Objective： To select the most efficient cyclin-dependent kinase （CDK） inhibitor for hepatoma cells Hep-3B and Hep-G2, and to investigate its effects on the proliferation and apoptosis of hepatoma cells. Methods： The inhibition rates of 10 kinds of CDK inhibitors on the proliferation of hepatoma Hep-3B and Hep-G2 cells were measured by CCK-8 assay, then the most efficient CDK inhibitor was selected. The half maximal inhibitory concentration （ICs0） values of CDK inhibitor in Hep-3B and Hep-G2 cells were detected by CCK-8 assay. The cell cycle distributions and apoptotic rates of Hep-3B and Hep-G2 cells after treatment with CDK inhibitor were detected by FCM. Furthermore, the expression levels of cell proliferation- and apoptosis-related proteins were detected by Western blotting. Results： The inhibitory rates of Hep-3B and Hep-G2 cells were both above 80% after treatment with 5 μmol/L BMS-265246 （No.6 CDK inhibitor） for 48 h, and the IC50 values were 2.84 and 1.73 μmol/L respectively. After treatment with 5 μmol/L BMS-265246 for 24 h, the proportions of Hep-3B and Hep-G2 cells in G1 phase increased significantly （both P 〈 0.05）, the phosphorylation of Rb and the expression of transcription factor E2F-targeted protein c-Myc were significantly down-regulated （both P 〈 0.01）. The apoptotic rates of Hep-3B and Hep-G2 cells after 5 μmol/L BMS-265246 treatment for 24 h and 48 h were significantly increased （all P 〈 0.05）. Furthermore, the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-xL in BMS-265246-treated group were significantly decreased （both P 〈 0.05）, while the expression levels of pro-apoptotic proteins Bax and Bak were significantly increased （both P 〈 0.05）. The cleaved caspase-3 and poly ADP-ribose polymerase （PARP） were found in Hep-3B and Hep-G2 cells after BMS-265246 treatment. Conclusion： CDK inhibitor BMS-265246 can induce apoptosis and inhibit proliferation of hepatoma Hep-3B and Hep-G2 cells in vitro, and its mechanism may be related to the anti-/ pro-apoptotic imbalance of Bcl-2 family members, the activation of caspase pathway, the dephosphorylation of Rb and the inhibition of E2F activity.