芹菜素对人NCI-H1650细胞的增殖抑制及诱导凋亡作用

Effect of Apigenin on Inhibiting Proliferation and Apoptosis of Human NCI-H1650 Cell Line

目的探讨芹菜素对人非小细胞肺癌NCI-H1650细胞的增殖抑制及诱导凋亡作用。方法体外培养NCI-H1650细胞,分别加入不同浓度的芹菜素(10,20,40,80μmol·L^(-1))处理NCI-H1650细胞,加入不含药物的等体积的RPMI-1640培养液为空白对照组。噻唑蓝(MTT)法测定不同浓度芹菜素对细胞增殖的抑制作用。Hoechst 33258细胞核染色法观察不同浓度的芹菜素作用后,细胞凋亡形态学的变化。流式细胞术Annexin V-异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染法检测不同浓度的芹菜素对细胞凋亡率的影响。Western blotting检测凋亡相关信号分子白血病-2相关X蛋白(Bax)、白血病-2(Bcl-2)的蛋白表达水平。结果 MTT法显示芹菜素对NCI-H1650细胞有增殖抑制作用,并呈一定的浓度和时间依赖性,差异有统计学意义(P<0.01)。Hoechst 33258细胞核染色法显示一定浓度依赖性的典型细胞凋亡特征,如核固缩和凋亡小体产生等。空白对照组,10,20,40,80μmol·L^(-1)的芹菜素作用NCI-H1650细胞48 h的凋亡率分别是(5.00±0.33)%,(18.05±4.67)%,(21.48±1.95)%,(43.24±1.11)%,(66.23±3.65)%,P<0.01。Western blotting结果显示,随着药物浓度升高Bax蛋白表达量增加,Bcl-2的表达水平随药物浓度升高而减少。结论芹菜素具有抑制及诱导非小细胞肺癌NCI-H1650细胞增殖、凋亡的作用,机制可能与肿瘤细胞内促凋亡相关蛋白Bax的表达增加、抗凋亡相关蛋白Bcl-2表达减少有关。

Objective To determine the effect of apigenin on inducing apoptosis and inhibiting proliferation of non-small cell lung cancer cell line NCI-H1650. Methods NCI-H1650 cell line was cultured routinely in vitro, with blank control group and different concentrations of apigenin （10, 20, 40, 80 μmol·L-1）. The blank control group was RPMI-1640 solution without apigenin. Cell proliferation was detected by MTT. Hoechst 33258 was applied to observe morphological changes of the apoptotic cells after treatment of different concentrations of apigenin. Flow cytometry AnnexinV-FITC/PI double staining method was used to determine cell apoptosis rate. The expression levels of apoptosis-related signaling molecules Bax and Bcl-2 protein were performed by Western blotting. Results MTT showed that apigenin inhibited proliferation of NCI-H1650 cell line in a concentration-and time-dependent manner （P〈0.01）.Hoechst 33258 nuclear staining showed typical characteristics of apoptosis in certain concentration-dependent manner, such as nuclear condensation and appearance of apoptotic bodies. In addition, the results of flow cytometry staining indicated that the apoptotic rate of NCI-H1650 cells cultured with blank control group and different concentrations of apigenin （10, 20, 40, 80 μmol·L-1） for 48 h was （5.00±0.33）%, （18.05±4.67）%, （21.48± 1.95）%, （43.24±1.11）%, （66.23±3.65）%, respectively （P〈0.01）.Western blotting results showed that the expression levels of Bax increased with increasing of the drug concentration, but Bcl-2 decreased with increasing of the drug concentration. Conclusion Apigenin can inhibit the proliferation and induce apoptosis of NCI-H1650 cells in non-small cell lung cancer. Mechanisms may be related to increase of the expression of apoptosis related protein Bax and decrease of the expression of anti apoptosis protein Bcl-2.