蛋白激酶CK2抑制剂对H460细胞内活性氧水平及DNA双链断裂影响

Effect of a protein kinase CK2 inhibitor on intracellular levels of reactive oxygen species and DNA double-stand break in H460 cells

目的：观察蛋白激酶CK2抑制剂对人肺癌细胞系H460细胞内活性氧水平及DNA双链断裂损伤的影响。方法使用终浓度为0、12.5、25.0、50.0μmol／L的蛋白激酶CK2抑制剂醌茜素分别作用H460细胞24 h，检测CK2各亚基蛋白及mRNA水平变化。不同浓度梯度醌茜素作用4、24 h后，采用流式细胞仪检测H460细胞内活性氧水平变化。通过免疫荧光法检测CK2抑制剂对H460细胞γ.H2 AX表达的影响，并计算细胞内γ.H2 AX焦点的平均值。采用方差分析和t检验。结果醌茜素对H460细胞 CK2各亚基蛋白或 mRNA 水平无明显影响（ CK2α，0μmol ∶12.5μmol／L， P＝0.966；0μmol／L ∶25μmol／L，P＝0.355；0μmol／L ∶50μmol／L，P＝0.864， CK2α’，0μmol／L ∶12.5μmol／L，P＝0.409；0μmol／L ∶25μmol／L， P＝0.833；0μmol／L ∶50μmol／L， P＝0.0．746， CK2β，0μmol／L ∶12.5μmol／L， P＝0.532；0μmol／L ∶25μmol／L， P＝0.830；0μmol／L ∶50μmol／L， P＝0.061）。随着醌茜素浓度增加及作用时间延长，H460细胞内活性氧水平明显升高。不同浓度醌茜素可增加细胞内γ.H2 AX焦点数，并呈剂量和时间依赖性（醌茜素作用时间4 h或24 h，0μmol／L ∶12.5μmol／L，12.5μmol／L ∶25μmol／L，25μmol／L ∶50μmol／L，P均＝0.000，浓度为12.5μmol／L，25μmol／L或50μmol／L，4 h ∶24 h，P均＝0.000）。结论醌茜素可通过抑制蛋白激酶CK2活性增加肺癌H460细胞内活性氧水平及DNA双链断裂损伤，该研究为醌茜素作为一种有潜力的肺癌放射增敏剂提供了理论基础。

Objective To evaluate the effect of a protein kinase CK2 inhibitor on intracellular levels of reactive oxygen species and DNA double.stand break in human non.small cell lung cancer H460 cells. Methods H460 cells were exposed to 0, 12.5, 25.0, and 50.0μmol/L quinalizarin, a specific inhibitor of protein kinase CK2, for 24 hours. The changes in protein and mRNA levels of CK2 subunits were measured. Flow cytometry was used to measure changes in the intracellular levels of reactive oxygen species in H460 cells after 4 or 24 hours of quinalizarin treatment. Immunofluorescence assays were performed to determine the effect of the CK2 inhibitor onγ.H2 AX expression and the average fluorescent number ofγ.H2 AX foci in H460 cells. Comparison was made by analysis of variance and t test. Results There were no significant differences in protein or mRNA levels of CK2 subunits in H460 cells after quinalizarin treatment （ CK2α,0μmol vs. 12.5 μmol/L, P=0.966;0 μmol/L vs. 25 μmol/L, P=0.355;0 μmol/L vs. 50 μmol/L, P=0.864, CK2α’ , 0 μmol/L vs. 12.5μmol/L,P=0.409;0μmol/L vs. 25μmol/L,P=0.833;0μmol/L vs. 50 μmol/L, P=0.0. 746, CK2β, 0 μmol/L vs. 12.5 μmol/L, P=0.532;0 μmol/L vs. 25 μmol/L, P=0.830;0 μmol/L vs. 50 μmol/L, P= 0.061 ） . The intracellular levels of reactive oxygen species were substantially elevated in H460 cells with the increase in quinalizarin concentration and treatment time. Different concentrations of quinalizarin resulted in dose.and time.dependent increases in the numbers of γ.H2 AX foci after 4 and 24 hours of treatment （ treated by Quianlizarin for 4 or 24 h, 0 μmol/L vs. 12.5μmol/L,12.5 μmol/L vs. 25 μmol/L, 25 μmol/L vs. 50 μmol/L, all P=0.000, concentration is 12.5μmol/L,25 μmol/L or 50 μmol/L, 4 h vs. 24 h, all all P=0.000 ） . Conclusions Quinalizarin can increase the intracellular levels of reactive oxygen species and DNA double.stand break in H460 cells by inhibition of protein kinase CK2 activity. This study provides a theoretical basis for using quinalizarin as a potential radiosensitizer for lung cancer.