华蟾素联合阿霉素对脑胶质瘤细胞增殖、体外侵袭能力的影响及机制研究

Effects and mechanism of cinobufacin combined with adriamycin on proliferation and invasion of glioma cells in vitro

目的:研究华蟾素联合阿霉素(Dox)作用于脑胶质瘤U251细胞对其体外侵袭能力的影响。方法:将脑胶质瘤U251细胞株消化处理成3×10^(4)/ml的悬液,接种于96孔板中,分四组。阿霉素组分别加入0.10、0.25、0.50、1.00、2.00 ng/ml的Dox;华蟾素组分别加入5.00、10.00、15.00、20.00、25.00μg/ml的华蟾素;联合组加入0.10、0.50、2.00 ng/ml的阿霉素和10.00μg/ml的华蟾素;对照组以同剂量RPMI-1640培养液替换。各组分别培养24、48、72 h,四甲基偶氮唑蓝比色法(MTT)法检测各组不同干预下脑胶质瘤U251细胞增殖抑制力的变化;逆转录-聚合酶链反应(RT-PCR)和蛋白免疫印迹(Western blot)法检测在不同干预下桩蛋白(Paxillin)、局部黏附斑激酶(FAK)和基质金属蛋白酶2(MMP-2)在各组细胞中的表达差异;Boyden chamber小室体外侵袭实验检测华蟾素联合阿霉素对脑胶质瘤U251细胞侵袭、迁移能力的影响。结果:阿霉素、华蟾素单药组、联合组均能抑制脑胶质瘤U251细胞增殖,但阿霉素达到一定浓度后抑制脑胶质瘤U251细胞的增殖效果欠佳,华蟾素联合阿霉素组对脑胶质瘤U251细胞的增殖抑制作用强于各单药作用组,且显示与药物浓度、作用时间呈正相关性。联合组对脑胶质瘤U251细胞的抑制增殖效果最显著。联合组中Paxillin、FAK和MMP-2的基因和蛋白表达水平明显下调,与其它单药组比较,差异具有统计学意义(均P<0.05);Boyden chamber小室体外侵袭实验结果显示,对照组穿膜细胞数最多,细胞迁移能力最强,且显著高于阿霉素、华蟾素单药组和联合组的穿膜细胞数,组间比较差异也具有统计学意义(均P<0.05)。结论:华蟾素联合阿霉素可以减弱脑胶质瘤U251细胞的体外侵袭能力,可能与Paxillin、FAK和MMP-2的基因、蛋白表达降低密切相关,这为脑胶质瘤U251细胞联合化疗和靶向治疗提供可行的实验理论依据。

Objective:To investigate the effects and mechanism of cinobufacin combined with adriamycin(Dox)on proliferation and invasion of glioma U251 cells in vitro.Methods:Glioma U251 Cell line was digested into 3×10^(4)/ml suspension,and was inoculated into 96 well plates,and was divided into four groups.The Dox group was added with Dox of 0.10,0.25,0.50,1.00,2.00 ng/ml,respectively.The cinobufacin group was added with 5.00,10.00,15.00,20.00,25.00μg/ml cinobufacin,respectively.The combined group was added with 0.10,0.50,2.00 ng/ml Dox and 10.00μg/ml cinobufagin.The control group was replaced with the same dose of RPMI-1640 culture medium.Each group was cultured for 24,48 and 72 hours respectively.The changes of proliferation inhibition of glioma U251 cells under different interventions were detected by MTT.RT-PCR and Western blot were used to detect the expression differences of Paxilin,focal adhesion kinase(FAK)and matrix metalloproteinase-2(MMP-2)in each group under different interventions.Boyden chamber invasion experiment in vitro was used to detect the effect of cinobufagin combined with Dox on the invasion and migration of glioma U251 cells.Results:Dox group,cinobufacin group and combined group could inhibit the proliferation of glioma U251 cells,but the effect of Dox on the proliferation of glioma U251 cells was not good after reaching a certain concentration.The inhibitory effect of combined group on the proliferation of glioma U251 cells was stronger than that of each single drug group,and showed a positive correlation with drug concentration and action time.The combined group had the most significant inhibitory effect on the proliferation of glioma U251 cells.The gene and protein expression levels of paxillin,FAK and MMP-2 in combined group were significantly lower than those in other single drug groups(all P<0.05).The results of Boyden chamber invasion experiment in vitro showed that the control group had the largest number of transmembrane cells and the strongest cell migration ability,which was significantly higher than that of Dox group,cinobufacin group and combined group(all P<0.05).Conclusion:Cinobufacin combined with Dox can reduce the invasion ability of gliomain U251 cells in vitro,which may be closely related to the decreased expression of Paxillin,FAK and MMP-2 genes and proteins,which provides a feasible experimental theoretical basis for combined chemotherapy and targeted therapy of glioma.