JNK通路参与曲古抑菌素A诱导的胰腺癌PANC-1细胞凋亡  

Role of JNK pathway in trichostatin A induced apoptosis of pancreatic carcinoma cell line PANC-1

作　　者：王本泉[1] 张玲[1] 黄雪[1] 陈宗静[1] 白永恒[1] 吴存造[2] 周蒙滔[1] 陈必成[1,2] 

机构地区：[1]温州医科大学附属第一医院外科实验室,浙江温州325000 [2]温州医科大学附属第一医院移植中心,浙江温州325000

出　　处：《中国药理学与毒理学杂志》2013年第5期801-807,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：温州市科技局项目(Y20090028);浙江省教育厅重中之重外科学项目~~

摘　　要：目的研究JNK通路在曲古抑菌素A(TSA)诱导人胰腺癌PANC-1细胞凋亡过程中的激活情况,并探讨其机制。方法 PANC-1细胞经TSA 1.0μmol·L-1与JNK抑制剂(SP600125)20μmol·L-1单独或联合处理24h后,分别用CCK-8法和Hoechst33258染色法检测对PANC-1细胞的生长和细胞凋亡的作用;荧光定量PCR检测c-Jun,c-fos,Elk1,bax和bcl-2 mRNA表达;Western蛋白质印迹法检测p-c-Jun、p-JNK、bax、活性胱天蛋白酶8、bcl-2和胱天蛋白酶3蛋白表达。结果 CCK-8结果显示,TSA单独处理,PANC-1细胞的存活率随着TSA浓度的增加而降低,并具有一定的量效关系(r=0.9564,P<0.05)。TSA+SP600125组细胞存活率较TSA单独处理明显增加(P<0.05)。Hoechst33258染色显示,正常对照组、TSA组和TSA+SP600125组凋亡率分别为(1.8±0.4)%,(6.2±1.4)%和(3.9±0.9)%,组间差异具有明显的统计学意义(P<0.05,24h)。与TSA组比较,TSA+SP600125组促凋亡基因bax以及JNK通路相关基因c-Jun和c-fos mRNA表达明显降低,Elk1mRNA表达降低,差异显著(P<0.05);而抗凋亡基因bcl-2mRNA表达明显升高(P<0.05)。TSA作用PANC-1细胞1h后激活JNK通路的相关蛋白c-Jun和JNK,并在2h达到最高峰。与TSA组比较,TSA+SP600125组bax及活性胱天蛋白酶8蛋白的表达明显降低(P<0.05),bcl-2和胱天蛋白酶3蛋白的表达明显升高(P<0.05)。结论激活JNK通路是TSA诱导PANC细胞凋亡的重要机制之一,可能是通过影响线粒体凋亡途径发挥作用。OBJECTIVE To investigate the role of c-Jun N-terminal kinase （JNK） pathway in the apoptosis of pancreatic carcinoma PANC-1 cell lines induced by trichostatin A （TSA） and the possible mechanism. METHODS PANC-1 cells were treated with TSA 1.0 μmol·L^-1 and/or combined with JNK inhibitor （SP600125） 20 μmol·L^-1 for 24 h, respectively. Cell viability and apoptosis were measured by CCK-8 assay and Hoechst33258 staining in vitro. RT-PCR and Western blotting were used to analyze the mRNA expression of c-Jun, c-fos, Elk1, bax and bcl-2 and the protein expression of p-c-Jun, p-JNK, Bax, activated caspase 8, bcl-2 and caspase 3 associated with apoptosis and JNK pathway, respectively. RESULTS After the treatment with TSA 1 iJmol.L-1, PANC-1 cell viability decreased with the increase in TSA concentration in a concentration-dependent manner （r=0.9564, P〈0.05）. However, the cell viability of combination group increased significantly compared with TSA group. PANC-1 cell viability increased with the concentration of JNK inhibitor, also in a concentration-dependent manner. Evidence from Hoechst33258 staining showed that the rate of PANC-1 cell apoptosis in normal control group, TSA group, TSA combined with JNK inhibitor group was （ 1.83 ± 0.45） %, （6.15 ± 1.42） % and （3.9 ±0.86）%, respectively （P 〈 0.05）. Compared with TSA group, the mRNA expression of pro-ap- optotic bax, c-Jun, c-los and Elk1 was negative-regulated in combination group（P〈0.05）, while antiapoptotic bcl-2 mRNA was obviously positive-regulated（ P 〈0.05）. Following treatment with TSA, p-JNK and p-c-Jun proteins were positive-regulated at 1 h and reached the peak at 2 h, Compared with TSA group, the level of Bax and activated caspase 8 expression was reduced significantly （ P 〈 0.05 ） in combination group, but the level of bcl-2 and caspase 3 expression increased significantly（ P 〈 0.05）. CONCLUSION JNK pathway plays an important role in TSA-induced apoptosis, and the mechanism may be related to mito

关 键 词：胰腺癌 曲古抑菌素A JNK通路 细胞凋亡 

分 类 号：R363[医药卫生—病理学]