蛋白激酶D抑制剂SD-208对非小细胞肺癌细胞增殖及凋亡的影响

Effect of protein kinase D inhibitor SD-208 on the proliferation and apoptosis of non-small cell lung cancer cells

目的探讨蛋白激酶D抑制剂SD-208对非小细胞肺癌细胞增殖、凋亡及细胞周期的影响。方法采用5、10、20、30μmol/L SD-208处理非小细胞肺癌细胞A549(设不加SD-208仅添加完全培养基组为对照组),分别在24、48、72 h 3个不同刺激时间点应用四甲基偶氮唑盐比色(MTT)法检测A549细胞的吸光值并计算增殖抑制率;膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)和碘化丙啶(PI)双染色法结合流式细胞术检测不同浓度SD-208处理A549细胞24、48 h后的细胞凋亡情况,PI染色法流式细胞仪检测分析不同浓度SD-208处理A549细胞48 h后细胞周期时相分布情况;Western blotting检测不同浓度SD-208处理48 h后的细胞周期蛋白A(Cyclin A)、Cyclin D1、Cyclin E、细胞周期蛋白依赖性激酶4(CDK4)和p16蛋白的表达水平。结果 SD-208可抑制A549细胞的增殖,且呈剂量和时间依赖性(P<0.05);SD-208处理后A549细胞的凋亡率及G0/G1期细胞比例均高于对照组,而S、G2/M期细胞比例均低于对照组,各浓度间的差异均有统计学意义(P<0.05);与对照组比较,SD-208处理后的Cyclin D1和CDK4蛋白水平降低,p16蛋白水平升高,差异均有统计学意义(P<0.05)。SD-208处理对A549细胞Cyclin A和Cyclin E蛋白水平无影响,与对照组比较差异无统计学意义(P>0.05)。结论 SD-208可抑制A549细胞增殖并诱导细胞凋亡及G0/G1期阻滞,其机制可能与其影响细胞周期及相关调控蛋白水平有关。

Objective To investigate the effect of protein kinase D inhibitor SD-208 on the proliferation, apoptosis and cell cycle of non-small cell lung cancer cells. Methods The A549 cells were treated with different concentrations of SD-208 （5, 10, 20, 30 μmol/L） , and the cells treated with only complete culture was used as the control group. MTT method was used to detect the absor- banee of A549 cells at 24, 48, and 72 h after treatment and the proliferation inhibition rates were calculated accordingly. Staining with Annexin V-fluorescein isothiocyanate （ Annexin V-FITC） and propidium iodide （PI） was employed to measure the apoptotie rates at 24 and 48 h via flow eytometry. The distribution of cell cycle phase was measured by PI staining after treatment with SD-208 for 48 h. Ex- pression levels of Cyclin A, Cyclin D1, Cyclin E, cyclin dependent kinase 4 （CDK4） and p16 protein were detected by Western blot- ting. Results Compared with the control group, SD-208 could inhibit the proliferation of A549 cells （P〈0. 05 ） , and the effect was exhibited in a dose- and time-dependent manner. As for SD-208 treated groups, apoptotic rates and percentages of G0/Gi phase cells were significantly higher than control group, and the proportion of S and G2/M cells were lower than those in control group （ P〈0. 05 ）. Compared with the control group, the levels of CDK4 and Cyclin D1 protein were decreased, and the level of p16 protein was increased after SD-208 treatment with statistical significance （ P〈0.05 ）. SD-208 treatment had no effect on the level of Cyelin A and Cyclin E in A549 cells （P〉0.05）. Conclusion SD-208 could inhibit the proliferation of A549 cells and induce cell apoptosis and G0/GI phase arrest, which may be related to its effect on cell cycle regulatory proteins.