曲古霉素A联合环巴胺对胰腺癌PANC-1细胞凋亡的影响  

Effect of Combination Usage of TSA and CYA on Pancreatic Cancer PANC-1 Cell Apoptosis

作　　者：尤和谊[1] 刘彪[1] 王本泉[1] 邹乾[1] 张玲[1] 白永恒[2] 周蒙滔[1] 陈必成[1,2] 陈宗静[1] 

机构地区：[1]温州医学院附属第一医院普外科,浙江温州325000 [2]温州医学院附属第一医院临床实验诊断中心,浙江温州325000

出　　处：《中国药学杂志》2014年第11期971-977,共7页Chinese Pharmaceutical Journal

基　　金：温州市科技局项目(Y20090028);浙江省卫生厅资助项目(11-ZC24;2012-XK-A28)

摘　　要：目的本实验旨在研究组蛋白去乙酰化酶抑制剂曲古霉素A(TSA)联合Hedgehog(Hh)信号通路阻断剂环巴胺(CYA)干预对胰腺癌PANC-1细胞凋亡的影响并初步探讨其可能机制。方法采用CCK8方法检测曲古霉素A、环巴胺作用24 h对PANC-1细胞50%抑制浓度(IC50)。用Hochest 33258荧光染色观察曲古霉素A、环巴胺单独及联合处理PANC-1细胞24 h后凋亡;荧光定量聚合酶链式反应(Q-PCR)检测Hedgehog信号通路关键分子及凋亡相关基因mRNA表达的变化;蛋白免疫印迹法检测凋亡相关蛋白(Bcl-2、活化胱天蛋白酶-3,活化胱天蛋白酶-8,活化胱天蛋白酶-9)及Hedgehog通路关键分子蛋白(Gli1、Smo、Ptc-1)表达的改变;细胞免疫荧光标记法检测Gli1蛋白表达的变化。结果 Hochest 33258染色显示,曲古霉素A及环巴胺均可诱导胰腺癌PANC-1细胞凋亡,24 h IC50分别为(0.51±0.07)μmol·L-1和(33.6±2.3)μmol·L-1,两者联合指数为0.835,具有低度协同作用。曲古霉素A及环巴胺干预后Bcl-2 mRNA表达下降,Bax mRNA表达上调,尤以联合组明显。曲古霉素A 0.5μmol·L-1干预可抑制Gli1 mRNA的表达,联合环巴胺20μmol·L-1后对Hedgehog通路的抑制效应进一步增强,降低Gli1、Smo mRNA表达量,上调Gli3 mRNA的表达。Western印迹结果显示,曲古霉素A 0.5μmol·L-1、环巴胺20μmol·L-1单独及联合处理PANC-1后,凋亡相关蛋白胱天蛋白酶3、胱天蛋白酶8和胱天蛋白酶9活化程度进一步上升,凋亡抑制因子Bcl-2的表达明显下降,Hedgehog信号关键分子蛋白Gli1、Smo、Ptc-1表达均明显下降,以联合组最为明显。细胞免疫荧光结果也显示Gli1表达明显下降。结论曲古霉素A、环巴胺可协同抑制Hh通路,诱导胰腺癌PANC-1细胞的凋亡。OBJECTIVE To evaluate the apoptosis effect of HDAC inhibitor trichostatin A （TSA） combination with Hedgehog （Hh） signaling inhibitor cyclopamine （CYA） on the PANC-1 cells line. METHODS Hochest 33258 staining was used to measure apoptosis of PANC-1 cells treated with TSA and CYA respectively or cooperatively. The 24 h IC50 of PANC-1 cells line responding to TSA and CYA were detected by CCK8. The mRNA expression of the gene relative with apoptosis and Hh signaling were evaluated by SYBR GREEN based FQ-PCR （fluorescent quantization polymerase chain reaction）. The proteins associated with apoptosis （Bcl-2 and activated caspase-3, activated caspase-8, activated caspase-9） and Hh signalling pathway （Glil, Smo and Ptc-1 ） were measured by Western blotting （WB）. Glil was also detected by cell immunofluorescence. RESULTS TSA and CYA could induce apoptosis of PANC-1 cells by Hochest 33258 stain, respectively or combinatively. The 24 h IC50 of PANC-1 cells line treated with chemical were （0. 51 ±0. 07） μmol ·L^-1 （TSA） and （33.6 ±2. 3） μmol ·L^-1 （CYA）, respectively; the combination index （CI） of TSA and CYA on the PANC-1 is 0. 835. Combination usage of TSA and CYA caused more significantly decreased expression of Bcl-2 mRNA and increased Bax mRNA as compared with the TSA or CYA treatment. TSA （0. 5 μmol ·L^-1 ） inhibited the expression of Glil mRNA, TSA promoted the inhibition effect of CYA on Hh pathway, down-regulation of Glil, Smo mRNA and up-regulation of Gli3 mRNA. Western blotting showed that combination usage of TSA and CYA induced more significant activation of activated caspase-3, activated caspase- 8, activated caspase-9 and reduction of Bel-2, Glil, Smo and Ptc-1 expression than TSA or CYA treatment. Decreased Gli 1 also confirmed by immunofluorescenee. CONCLUSION Combination usage of TSA and CYA could induce PANC-1 cells line apoptosis and synergistically inhibit Hh pathway.

关 键 词：胰腺癌 曲古霉素A HEDGEHOG 细胞凋亡 环巴胺 

分 类 号：R965[医药卫生—药理学]