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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

王稣佳1,胡 敏2   

  1. 1 610064 成都 四川大学望江医院普内科2 450003 河南省人民医院检验科
  • 收稿日期:2015-10-01 修回日期:2015-12-21 出版日期:2016-02-29 发布日期:2016-02-29

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

WANG Sujia, HU Min.
  

  1. Department of Internal Medicine, Wangjiang Hospital of Sichuan University, Chengdu 610064, China
  • Received:2015-10-01 Revised:2015-12-21 Online:2016-02-29 Published:2016-02-29

摘要: 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。

Abstract:

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.

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