芒柄花黄素对非小细胞肺癌A549细胞增殖及上皮间质转化的抑制作用

摘要

Objective To explore the inhibitory effect of formononetin ( Form) on proliferation and epithelial mesenchymal tran⁃sition ( EMT) of non⁃small cell lung cancer A549 cells. Methods The A549 cells were cultured with Form ( a final concentration of 0. 1, 1, 10, 100μmol/L) . The cell proliferation was determined at 24, 48, 72 and 96 h post⁃treatment by MTT method in control group and From group. The Annexin V⁃FITC/PI double staining combined with flow cytometry and quantitative real⁃timepolymerase chain reac⁃tion were applied to measure the early⁃apoptotic and late⁃apoptotic rates and expression of apoptosis related genes at 48 h after treatment. Western blotting was used to detect the EMT related molecular markers, including E⁃cadherin, N⁃cadherin and Vimentin. The supernatant levels of fibronectin ( FN) were measured at 24, 48, 72 and 96 h by enzyme linked immunosorbent assay. Results Form exerted the in⁃hibitory effect on the proliferation of A549 cells in the range of 1⁃100 mol/L, and presented in a dose⁃and time⁃dependent manner. In ad⁃dition to 0. 1 mol/L at 24⁃48 h, the proliferation inhibiting rates of Form were higher than those of control group ( P<0. 05) . Compared with the control group, there were higher levels of early⁃apoptotic and late⁃apoptotic rates and mRNA levels of Bax and Bak but lower Bcl⁃2 mRNA levels in Form groups with statistical significance (P<0. 05). In response to Form, there were increased E⁃cadherin but de⁃creased N⁃cadherin, Vimentin and FN in comparison with control group at 48 h. Conclusion Form has a toxic effect on A549, which not only inhibit the proliferation and induce apoptosis. It may be related to the inhibition of the EMT process.%目的：探讨芒柄花黄素（ Form）对人非小细胞肺癌A549细胞增殖、凋亡及上皮间质转化（ EMT）的影响。方法采用终浓度为0．1、1、10、100μmol／L Form处理A549细胞，应用四甲基偶氮唑盐比色法测定对照组（不加药物）及不同浓度Form处理24、48、72和96 h的细胞增殖情况，分别采用Annexin V⁃FITC／PI双染联合流式细胞术和实时荧光定量聚合酶链反应检测不同浓度Form处理48 h的凋亡率及凋亡相关基因的表达情况，Western blotting检测各组处理48 h后的EMT相关分子标志物（ E⁃cadherin、N⁃cadherin及Vimentin）水平，采用酶联免疫吸附法测各组处理24、48、72及96 h的细胞上清液中纤连蛋白（ FN）水平。结果 Form在1～100μmol／L的范围内对A549细胞有增殖抑制作用，增殖抑制率呈浓度和时间依赖性，且除24～48 h 0．1μmol／L外，其余浓度及作用时间的增殖抑制率均高于对照组（ P＜0．05）；与对照组比较，Form处理48 h后的早期、晚期凋亡率及Bax、Bak的mRNA水平均升高，而Bcl⁃2水平降低，差异均有统计学意义（ P＜0．05），且呈剂量依赖性；与对照组比较，不同浓度Form处理48 h后的N⁃cadherin和Vimentin水平及上清液FN水平均降低，而E⁃cadherin水平升高，以上差异有统计学意义（ P＜0．05）。结论 Form对非小细胞肺癌A549细胞有毒性作用，不仅抑制其增殖还诱导凋亡，可能与抑制其EMT过程有关。