三羟基异黄酮联合放疗对人肺腺癌细胞增殖凋亡的影响

Effect of Genistein combined with radiotherapy on cell proliferation and apoptosis in pulmonary adenocarci noma A549 cells

目的观察三羟基异黄酮(Genistein)联合放疗对人肺腺癌细胞A549增殖凋亡及其Ape1/Ref1蛋白的影响。方法 1组单用0、10、30、60、90μmol/L的Genistein处理A549细胞48 h,另1组用同样浓度的Genistein预处理24 h后予以直线加速器放疗8 Gy,继续培养24 h后用噻唑蓝比色法(MTT)检测各组活细胞数,确定最适Genistein联合放疗的浓度。用流式细胞仪检测对照组、单纯放疗组(IR 8 Gy)、单纯Genistein组(GEN 30μmol/L)、Genistein+IR组(GEN30μmol/L+IR 8 Gy)的细胞凋亡;采用Western blot法检测各组细胞中Ape1/Ref1蛋白表达。结果 30、60、90μmol/L的Genistein联合放疗比单用相应浓度的Genistein处理细胞增殖抑制率高(P<0.05),但低浓度10μmol/L联合放疗与单纯放疗比较差异不明显[(26.67±1.26)%vs(24.70±0.72)%,P=0.102]。IR组、Genistein+IR组早期凋亡率较对照组明显增高[(30.88±7.63)%、(42.98±1.46)%vs(6.80±0.98)%,P<0.05],加药组较对照组无明显统计学差异[(12.96±4.99)%vs(6.80±0.98)%,P=0.143)];与对照组、单纯放疗组、单纯加药组比,Genistein+IR组中Ape1/Ref1的表达明显下调。结论三羟基异黄酮可增加放疗对A549细胞的增殖抑制作用,可能与减少Ape1/Ref1蛋白的表达有关。

Objective To examine the effect of Genistein combined with radiotherapy on the proliferation,apoptosis and Ape1/Ref1 protein expression of human pulmonary adenocarcinoma cell line A549.Methods A549 cells were divided into 4 groups including a control group（cells with no treatment）,a Genistein group（cells treated with Genistein in the doses of 0 μmol/L,10 μmol/L,30 μmol/L,60 μmol/L and 90 μmol/L,respectively,for 48 h）,an IR group（cells treated with a 8 Gy irradiation） and a Genistein ＋IR group（cells pretreated with the same doses of Genistein for 24 h and with a 8 Gy irradiation by a linear accelerator and continuously cultured for another 24 h）.The cell proliferation was examined by methyl thiazolyl tetrazolium（MTT） assay to determine a most suitable Genistein concentration combined with radiotherapy.Cell apoptosis of the 4 groups was observed by flow cytometry,and Ape1/Ref1 protein expression was measured by Western blotting.Results The inhibition rate of cell proliferation was higher in the treatment of 30 μmol/L,60 μmol/L and 90 μmol/L Genistein combined with irradiation than in the same doses of Genistein treatment alone（P0.05）,and the most suitable concentration of Genistein was 30 μmol/L.No significant difference was found between the cells treated with low concentration（10 μmol/L） of Genistein combined with irradiation and with irradiation alone [（26.67±1.26）% vs（24.70±0.72）%,P=0.102].The early apoptotic rates of the IR group and the Genistein＋IR group were significantly higher than that of the control group[（30.88±7.63）%,（42.98±1.46）% vs（6.80±0.98）%,respectively,P0.05],while there was no statistical difference between the control group and the Genistein group [（12.96±4.99）% vs（6.80±0.98）%,P=0.143].As compared with the Ape1/Ref1 protein expression of the control group,the IR group and the Genistein group,that of the Genistein＋IR group decreased significantly.Conclusion Genistein（30 μmol/L） can enhance the inhibition effect of radiotherapy on A549 cell proliferation through the reduction of Ape1/Ref1 protein expression.