摘要
目的研究雷酚萜甲醚(TME)在体外对人胃癌AGS细胞的增殖抑制和诱导凋亡作用。方法采用MTT法观察TME对人胃癌AGS细胞、正常人胃黏膜上皮细胞GES-1的增殖抑制作用;克隆形成实验观察细胞克隆的形成;光镜下及AO/EB染色观察细胞形态;流式细胞术检测细胞凋亡和细胞周期;JC-1染色和DCFH-DA荧光探针分别检测TME对AGS细胞线粒体膜电位的改变和活性氧产生的影响;Western blot检测凋亡蛋白caspase-3、caspase-8和Bcl-2、Bax表达情况,以及caspase广谱抑制剂z-VAD-fmk对caspase-3、caspase-8蛋白表达的影响。结果 TME可明显抑制人胃癌AGS细胞的增殖,并诱导其凋亡,作用48 h时IC50为23.85μmol·L-1,而对正常人胃黏膜上皮细胞GES-1的抑制作用明显低于AGS。TME能够抑制AGS细胞克隆形成,并使细胞出现明显的凋亡形态改变。Annexin V-FITC/PI双染实验表明,随TME剂量的增加,细胞凋亡百分数也增加。JC-1和DCFH-DA结果显示,TME使细胞内线粒体膜电位降低、细胞内活性氧水平增加。Western blot结果显示,TME增加了Bax/Bcl-2的比例,激活caspase-8和caspase-3,并且加入z-VAD-fmk后,caspase-3、caspase-8蛋白的表达降低。TME可使AGS细胞周期阻滞于G0/G1期。结论 TME能抑制人胃癌AGS细胞增殖和诱导凋亡,抗肿瘤作用机制与激活凋亡通路、影响细胞周期及Bcl-2蛋白家族相关。
Aim Toinvestigatetheeffectsoftriptonot-erpene methyl ether （ TME ） , a diterpene derived from the medicinal plant Triptergium wilfordii, on human gastric cancer AGS cell proliferation inhibition and ap-optosisinducedinvitro.Methods MTTassaywas used for screening tumor spectrum and detecting the vi-ability of AGS cells and normal human gastric epitheli-al cells GES-1 . Cell morphology was observed by light microscopy and AO / EB staining. Flow cytometry was used to detect cell apoptotic rate and cell cycle. JC-1 staining and fluorescence probe DCFH-DA were em-ployed to detect the changes of mitochondrial mem-brane potential and reactive oxygen species （ ROS ） . The effect of inhibiting AGS clonogenic survival was as-sayed by the method of plate clone formation. Western blot was used to analyse the expression of caspase-3 , caspase-8,Bcl-2andBax.Results MTTresults showed that TME exhibited significantly higher cytotox-icity to gastric cancer AGS cell line than to noncancer-ous cell line GES-1. IC50 for AGS of 48 h treatment was 23 . 85 μmol ＆#183; L-1 . TME significantly inhibited colony formation and caused morphological changes in AGS cells. Annexin V-FITC / PI double staining showed the apoptotic rate increased. DCFH-DA stai-ning showed TME resulted in an increase in intracellu-lar ROS levels. Mitochondrial membrane potential de-creased after TME treatment. Western blot results showed that TME increased the proportion of Bax /Bcl-2 , with the activation of caspase-8 and caspase-3 . The broad-spectrum caspase inhibitor z-VAD-fmk pre-treatment reduced the expression of caspase-8 and caspase-3. TME enabled AGS cell cycle arrest in G0/G1phase.Conclusion TMEpossessespotenttumor selected toxicity and can induce apoptosis of AGS cells through cell cycle arrest, which is associated with Bcl-2 protein family.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第8期1066-1072,共7页
Chinese Pharmacological Bulletin
基金
国家重点基础研究发展计划(973计划)资助项目(No2009CB522806)
国家科技部"重大新药创制"科技重大专项资助项目(No 2012ZX09501001-004)
