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

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、30 μmol／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。

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

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 absorbance 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 apoptotic rates at 24 and 48 h via flow cytometry. The distribution of cell cycle phase was measured by PI staining after treatment with SD-208 for 48 h. Expression levels of Cyclin A， Cyclin D1, Cyclin E，cyclin dependent kinase 4 （CDK4） and p16 protein were detected by Western blotting. 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／G1 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 Cyclin 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／G1 phase arrest， which may be related to its effect on cell cycle regulatory proteins.