曲古抑菌素A对细胞PANC-1株凋亡和肿瘤转移相关基因表达的影响  

Effect of trichostatin A on expression of apoptitosis-related genes and metastasis-related genes in human pancreatic cancer PANC-1 cells

作　　者：杨云秀[1] 胡孙宽[2] 叶星照[2] 白永恒[3] 王斯璐[3] 刘彪[1] 王本泉[1] 陈必成[3] 陈宗静[1] 

机构地区：[1]温州医学院附属第一医院肝胆胰外科,浙江温州325000 [2]温州医学院附属第一医院消化内科,浙江温州325000 [3]温州医学院附属第一医院外科实验室,浙江温州325000

出　　处：《中国药理学与毒理学杂志》2012年第5期624-629,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：温州市科技局项目(Y20090028);浙江省重中之重学科外科学项目~~

摘　　要：目的探讨曲古抑菌素A(TSA)诱导胰腺癌细胞PANC-1细胞凋亡机制。方法 TSA 0.1～0.6μmol.L-1培养PANC-1细胞0～48 h,MTT法检测细胞存活率并计算IC50。TSA 0.4μmol.L-1PANC-1培养0～48 h,Hoechst 33258染色观察细胞核形态变化。TSA 0.4μmol.L-1PANC-1培养0～12 h,检测胱天蛋白酶3活性。TSA 0.4μmol.L-1与PANC-1培养24 h,实时定量PCR检测c-myc,p53,Bcl-2,Bax,存活素,基质金属蛋白酶1(MMP1)和基质金属蛋白酶1组织抑制剂(TIMP-1)和Notch-1基因的表达。TSA 0.4和0.6μmol.L-1与PANC-1培养24 h,细胞免疫化学方法检测Notch-1蛋白的胞内活性形式NICD表达。结果TSA可以明显抑制PANC-1细胞增殖,12,24和48 h的IC50值分别为0.42,0.32和0.19μmol.L-1,具有量效(r=0.640,P=0.01)和时效(r=0.768,P=0.002)关系。Hoechst 33258染色结果表明,TSA 0.4μmol.L-1增加PANC-1细胞核的蓝色荧光并出现凋亡特征。TSA 0.4μmol.L-1作用4,8和12 h后,胱天蛋白酶3的活性分别为正常对照组的1.62±0.12,2.68±0.17和(3.92±0.23)倍。PCR结果显示,TSA 0.4μmol.L-1作用24 h后,p53,c-myc和存活素mRNA表达下降,分别为对照组的(18.3±5.1)%,(24.2±0.9)%和(15.8±1.0)%,Bcl-2和Notch-1 mRNA未见明显变化,而Bax,MMP1和TIMP-1 mRNA升高(P<0.05),Bcl-2/Bax比值降低到正常对照组的(13.0±2.8)%。Notch-1活性分子NICD明显升高(P<0.05)。结论TSA可通过线粒体途径诱导胰腺癌PANC-1细胞凋亡,而且使Notch-1激活,促进转移相关基因表达。OBJECTIVE To investigate the mechanism of trichostatin A（TSA） in inducing apoptosis in human pancreatic cancer PANC-1 cells and the genes involved in apoptosis.METHODSThe proliferation inhibition rate and IC50 of TSA were measured by MTT assay in vitro after PANC-1 cells were treated with TSA 0.1-0.6 μmol·L-1 for 12,24 and 48 h,respectively.Hoechst 33258 staining（TSA 0.4 μmol·L-1 for 0-48 h） and caspase 3 activity assay（TSA 0.4 μmol·L-1 for 0-12 h） were used to determine apoptosis.After the treatment with TSA 0.4 μmol·L-1 for 24 h,the expression of c-myc,p53,Bcl-2,Bax,survivin,matrix metalloproteinases（MMP1）,tissue inhibitor of matrix metalloproteinase-1（TIMP-1） and Notch-1 were evaluated by SYBR GREEN based RT-PCR.The cytoplasmic tail of Notch-1（NICD） in PANC-1 was semi-quantified by immunocytochemistry.RESULTS TSA could obviously inhibit proliferation of PANC-1 cells and the inhibition rate significantly increased in a concentration-and time-dependent manner.The IC50 was 0.42,0.32 and 0.19 μmol·L-1 after PANC-1 cells were treated with TSA for 12,24 and 48 h,respectively.TSA could promote apoptosis of PANC-1 cells.At 4,8 and 12 h of TSA 0.4 μmol·L-1 treatment,the activities of caspase 3 increased to 2.62±0.12,3.68±0.17,4.92±0.23 fold as compared to untreated PANC-1.Compared with normal control,the expression of p53,c-myc and survivin mRNA was down-regulated to（18.3±5.1）%,（24.2±0.9）% and（15.8±1.0）%,respectively（P0.05）,Bax,MMP1 and TIMP-1 up-regulated（P0.05）,and Bcl-2,Notch-1 were not affected.Significant increase of NICD in PANC-1 was detected after TSA 0.4 μmol·L-1 was treated for 24 h（P0.05）.CONCLUSION TSA inhibits pancreatic cancer cell line PANC-1 by inducing apoptosis through mitochondrial pathways,which can cause Notch-1 pathyway action and transcription of target genes.

关 键 词：曲古抑菌素A 胰腺癌 细胞凋亡 NOTCH通路 

分 类 号：R735.9[医药卫生—肿瘤]