摘要
目的研究木犀草素对胃癌细胞MGC803的增殖抑制作用及其机制。方法通过MTT法、克隆形成抑制实验观察木犀草素对胃癌细胞MGC803的增殖抑制作用,碘化丙啶(PI)单染色法检测细胞周期,AnnexinⅤ-PI双染法测定细胞凋亡,蛋白质印迹法检测Bcl-2家族蛋白、Caspase蛋白、周期及自噬相关蛋白的表达情况。结果 MTT法检测发现,木犀草素作用于MGC803细胞24、48和72h后,IC50分别为127.37、76.12、16.84μmol/L;木犀草素能抑制胃癌细胞MGC803的克隆形成。PI单染色法发现随着木犀草素浓度的增大,发生G2期阻滞的细胞比例增加。蛋白质印迹法的检测结果表明,随着木犀草素浓度的增加,CDK4、CDK6、CyclinE2表达减少,CyclinD1、CyclinD3和p-Wee1表达基本不变,CyclinA、CyclinB、Myt1、p-cdc2(Tyr15)表达增高。AnnexinⅤ-PI双染法发现随木犀草素浓度增加,细胞的早期凋亡率和晚期凋亡率均增加。蛋白质印迹法的检测结果表明,Mcl-1、Bcl-xL、Caspase-3、Caspase-8、Caspase-9蛋白表达随木犀草素浓度增加而减少,Bcl-2蛋白表达量变化不大,Bax蛋白表达量增加,因而引起Bcl-2/Bax、Mcl-1/Bax和Bcl-xL/Bax的比例下降;同时可见到Caspase-3和PARP出现了切割的条带;P62表达下降,Beclin-1、LC3Ⅱ表达增加。结论木犀草素使胃癌细胞MGC803发生G2期阻滞,诱导细胞自噬的发生并抑制细胞的生长,还通过线粒体途径诱导胃癌细胞MGC803凋亡。
Objective To investigate the inhibitory effect of luteolin against proliferation of gastric cancer cell line MGC803 and the related mechanism. Methods The inhibitory effect of luteolin against MCA2803 cell proliferation was evaluated by MTT assay and clone forming assay. The cell cycle changes were analyzed by the flow cytometry after staining propidium iodide （PI）. Annexin V-PI double staining was used to determine the apoptosis ratio of MGC803 cells. Bcl-2 family proteins, Caspase proteins, autophagy-associated and cell cycle-associated proteins were assessed by Western blotting analysis. Results MTT assay results showed that the IC.~0 of luteolin against MCX2803 cells for 24 h, 48 h and 72 h treatment were 127.37 μmol/L, 76.12 μmol/L, and 16. 84 μmol/L, respectively. Luteolin treatment also inhibited cell colony formation of MGC2803 cells. PI single staining flow cytometry found that cells were arrested in G2 phase with the increase of luteolin concentration. Western blotting results showed that, with the increase of luteolin concentration, CDK4, CDK6 and CyclinE2 protein expression was decreased; CyclinD1, CyclinD3 and p-Weel protein kept unchanged; and CyclinA, CyclinB, Mytl and pcdc2 （Tyr15） protein expression was decreased. Flow cytometry Annexin V-FITC/pI revealed increase of early and late apoptosis with the increase of luteolin concentration. Western blotting analysis showed that Mel-1, Bcl-xi., Caspase-3, Caspase- 8 and Caspase-9 protein expression was decreased with the increase of luteolin concentration, with slightly changed Bcl-2 expression and increased Bax expression, which led to decreased Bcl-2/Bax, Mcl-1/Bax and Bcl-xL/Bax ratios. At the same time, Caspase-3 and PARP also appeared in the strip cutting. The expression of P62 was decreased and expression of Beclin-1 and LC3 Ⅱ was increased. Conclusion Luteolin can arrest gastric cancer MCA2803 cells at G2 phase, inducing autophagy and inhibiting cell growth. Luteolin can also induce apoptosis of MCA2803 cells via the mitochondrial p
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2016年第3期302-308,共7页
Academic Journal of Second Military Medical University
基金
广东省自然科学基金(2014A030313356)
广东省科技计划项目(2012B060300020)
华南肿瘤学国家重点实验室开放课题(HN2015-04)
暨南大学科研培育与创新基金跃升计划项目(116154240)~~
