Rho激酶抑制剂Y27632对非小细胞肺癌增殖、凋亡及侵袭转移的影响

Effects of Rho kinase inhibitor Y27632 on proliferation,apoptosis and invasion of non-small cell lung cancer

目的探讨Rho激酶抑制剂Y27632对非小细胞肺癌细胞增殖、凋亡及侵袭转移的影响。方法采用5、10、20、40、80μmol/L Y27632处理非小细胞肺癌细胞A549后,设不加药物的为对照组。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法(MTT法)检测A549细胞的吸光度并计算增殖抑制率和半数抑制浓度(IC_(50)),膜联蛋白-增强型绿色荧光蛋白(Annexin-EGFP)/碘化丙啶(PI)双染流式细胞术检测Y27632处理24、48 h后的细胞凋亡情况,PI单染流式细胞术检测Y27632处理48 h后细胞周期各时相(G_0/G_1、S和G_2/M期)分布情况,Transwell试验检测Y27632处理48 h后的穿膜细胞数。结果 Y27632可抑制A549的增殖(与对照组比较,P<0.05),呈剂量和时间依赖性,且IC50值随作用时间的延长而降低;与对照组比较,Y27632处理后的凋亡率、G_0/G_1期细胞比例均升高,而S、G_2/M期细胞比例均降低,各浓度间的差异均有统计学意义(P<0.05);Transwell实验发现Y27632可抑制A549细胞的侵袭,如0、5、10、20、40、80μmol/L Y27632处理后的穿膜细胞数依次为(78.1±6.0)、(62.3±5.7)、(51.7±5.2)、(42.3±6.9)、(36.4±5.3)和(22.8±4.6)个,差异均有统计学意义(P<0.05)。结论 Y27632可抑制A549细胞的增殖并诱导凋亡及G_0/G_1期阻滞,同时可抑制细胞侵袭,在肺癌治疗上有一定价值。

Objective To investigate the effect of Rho kinase inhibitor Y27632 on cell proliferation, apoptosis and invasion and metastasis of non-small cell lung cancer. Methods The non-small cell lung cancer cells A549 were treated with different concen-trations of Y27632 （ 5, 10, 20, 40, 80μmol/L） and the cells without treatment were set as the control group. The absorbance of A549 cells was detected by MTT method at 24, 48, 72 h after stimulation, and the inhibitory rates of proliferation and half inhibitory concen-trations （ IC50 ） were calculated accordingly. The cell apoptotic rates were measured at 24 and 48 h by Annexin-enhanced green fluores-cent protein （Annexin-EGFP）/iodinated propidium （PI） double staining via flow cytometry. The distribution of cell cycle, including G0/G1 , S and G2/M, were detected by PI single staining with flow cytometry. The number of cells penetrating the membrane was de-tected by Transwell test. Results Y27632 can inhibit the proliferation of A549 （ compared with the control group P〈0？05） in a dose-and time-dependence manner, and the IC50 value decreased with the prolongation of the time. Compared with the control group, the ap-optotic rates and G0/G1 phase cell proportions were increased, and the proportions of S and G2/M cells were decreased, and the differ-ences among different concentrations were statistically significant （ P〈0？05） . Transwell experiments showed that Y27632 could inhibit the invasion of A549 cells. For instance, the number of cells penetrating the membrane were （78？1±6）, （62？3±5？7）, （51？7±5？2）, （42？3±6？9）, （36？4±5？3） and （22？8±4？6） for treatment with 0, 5, 10, 20, 40, 80 mol/L Y27632, respectively. Conclusion In addition to the inhibition effect on cell invasion, Y27632 can inhibit the proliferation of A549 cells and induce apoptosis and G0/G1 ar-rest, having a certain value in the treatment of lung cancer.