摘要
目的:探讨水飞蓟宾对胰腺癌As PC-1细胞的增殖抑制作用及其作用机制.方法:MTT法和克隆形成抑制实验观察水飞蓟宾对人胰腺癌As PC-1细胞的增殖抑制作用,碘化丙锭(PI)单染色检测细胞周期改变,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡水平,Western blotting检测细胞周期及细胞凋亡相关蛋白的表达.结果:不同浓度的水飞蓟宾对胰腺癌As PC-1细胞的生长均有抑制作用,且呈剂量-效应和时间-效应关系(P<0.05),水飞蓟宾作用于As PC-1细胞48、72 h的IC50浓度分别为224.20、87.25μmol/L;克隆形成抑制实验显示,随着水飞蓟宾浓度增加,As PC-1细胞克隆形成逐渐减少.细胞周期检测结果显示,随着水飞蓟宾浓度的增加,胰腺癌As PC-1细胞出现明显G1期阻滞;水飞蓟宾处理组细胞的周期蛋白Cyclin D1、Cyclin E2、Cyclin A、Cyclin B1表达下降,细胞周期蛋白激酶CDK4、CDK6表达不变,细胞周期素依赖性蛋白激酶抑制蛋白P15INK4B、P21WAF1/CIP1表达升高,与流式检测的结果相一致.不同浓度水飞蓟宾作用48 h后,出现明显的凋亡细胞群;同时发现Caspase-9、Caspase-3活化降解,Caspase3下游效应蛋白PARP出现切割条带.JNK蛋白表达增加并磷酸化活化,Bcl-2蛋白家族中抗凋亡蛋白Bcl-2、Bcl-x L、Mcl-1表达明显降低,促凋亡蛋白Bax表达基本不变,BH3-only蛋白Bclxs、Bid、Bim表达增加.结论:水飞蓟宾明显抑制胰腺癌细胞增殖,通过诱导P15INK4B、P21WAF1/CIP1表达阻滞细胞周期在G1期,并通过诱导JNK活化激活线粒体细胞凋亡途径,进而诱导胰腺癌As PC-1细胞凋亡.
Aim：To investigate the inhibitory effects of Silibinin on pancreatic cancer AsPC-1 cells and the possible mechanism.Methods：MTT and cell clone formation inhibitory assay were used to in- 〈br〉 vestigate the inhibitory effects of Silibinin on human pancreatic cancer AsPC-1 cells.Cell cycle distribu-tion and cell apoptosis were determined by flow cytometry staining with propidium iodide （PI）or AnnexinⅤ-FITC and PI respectively.The expressions of cell cycle and apoptosis related proteins were analyzed by Western blotting.Results：MTT results showed that Silibinin inhibited pancreatic cancer AsPC-1 cells′proliferation in dose and time-dependent manner（P 〈0.05）.The IC50 of Silibinin against pancreat-ic cancer AsPC-1 cells for 48 hours and 72 hours was 224.20 μmol /L and 87.25 μmol /L,respectively. Cell colony formation inhibition assay showed that the number of cell clones were decreased with the in-creasing of Silibinin′s concentration.PI staining analysis showed that cell cycle was arrested at G1 phase. Western blotting assay showed that the levels of Cyclins （D1,E2,A,B1）decreased,without changes in cyclin-dependent kinases （CDK4 and CDK6 ）,but cyclin-dependent kinase inhibitors （P15 INK4B , P21 WAF1 /CIP1 ）increased,which was in consistence with the results of flow cytometric analysis.Annexin V-FITC /PI staining analysis also showed a dose-dependent effect on apoptotic cells when the pancreatic cancer cells were incubated with Silibinin for 48 hrs.Caspase-9,Caspase-3 were cleaved,and the cleaved bands of PARP were detected.The expression of JNK and phospho-JNK were found to be in-creased,while the anti-apoptotic proteins of Bcl-2,Bcl-xL and Mcl-1 were decreased,and pro-apoptotic proteins Bax was not changed.The BH-3 only proteins,Bcl-xs,Bid and Bim,were found to be in-creased by Western blotting.Conclusion：Silibinin inhibits proliferation of pancreatic cancer AsPC-1 cells,induces G1 phase arrest via upregulating P15 INK4B and P21 WAF1 /CIP1 ,and induces cell apoptosis through
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2015年第1期21-28,共8页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
广东省科技计划项目(2011B031800012)
广东省医学基金项目(A2013338)
关键词
水飞蓟宾
胰腺癌
AsPC
-1
细胞
细胞周期
凋亡
Silibinin
pancreatic cancer AsPC-1 cells
cell cycle distribution
apoptosis
