摘要
目的研究脱氢枞胺对氟苯甲醛(DHAA-F)对人肝癌Hep G2细胞存活的影响,探讨其诱导细胞凋亡作用机制。方法用不同浓度DHAA-F处理Hep G2细胞24,48和72 h,CCK-8法检测细胞存活;DHAA-F20,40和80μmol·L^(-1)处理Hep G2细胞24 h,荧光显微镜观察细胞形态的变化,Annexin V-FITC/PI双染检测细胞凋亡,Western蛋白印迹法检测凋亡相关蛋白BCL-2、BAX、活化的胱天蛋白酶9和胱天蛋白酶3蛋白表达水平,以及丝裂原激活蛋白激酶(MAPK)家族中ERK,JNK和P38蛋白的表达。结果与细胞对照组比较,DHAA-F可显著抑制细胞存活(P<0.01),24,48和72 h的IC50值分别为56.8±4.4,40.2±3.4和24.2±2.4μmol·L^(-1);DHAA-F 20,40和80μmol·L^(-1)作用24 h后,核固缩程度加深,PI染色增多,细胞凋亡率明显增加(P<0.01),由细胞对照组的(6.4±0.6)%分别增加至(12.3±1.7)%,(28.8±3.2)%和(61.8±4.6)%;DHAA-F可以增加Hep G2细胞中JNK和P38蛋白的磷酸化(P<0.01),引起BCL-2表达下调(P<0.01)、BAX及活化的胱天蛋白酶9和胱天蛋白酶3表达上调(P<0.01);与DHAA-F组相比,P38MAPK抑制剂SB203580和JNK抑制剂SP600125可逆转DHAA-F引起的BCL-2表达下调、BAX表达上调和胱天蛋白酶3的活化(P<0.01)。结论 DHAA-F可通过激活JNK/P38通路诱导人肝癌Hep G2细胞发生凋亡。
OBJECTIVE To investigate the effects and mechanisms of dehydroabietylamine-fluorobenzaldehyde（DHAA-F）on cell proliferation and apoptosis in human hepatocellular carcinoma Hep G2 cells. METHODS Survival of Hep G2 cells treated with DHAA-F 12.5,25,50,100 and 200 μmol·L^（-1）was measured with Cell Counting Kit- 8 assay. Hep G2 cells were treated with DHAA- F 20,40 and80 μmol·L^（-1）for 24 h,the morphological features were observed under the fluorescence microscope.Apoptosis was determined by Annexin V-FITC staining and PI labeling,white protein expressions of BCL-2,BAX,cleaved caspase-9/3,ERK,JNK,and P38 MAPK were measured by Western blotting analysis. Furthermore. Hep G2 cells were treated with SP600125（an inhibitor of JNK）or SB203580（an inhibitor of P38）,and then treated with DHAA-F. JNK/P38 pathway was measured to confirm its involvement in the DHAA- F- induced apoptosis. RESULTS Compared with control group,DHAA- F could obviously inhibit the proliferation of Hep G2 cells（P〈0.01）. IC50 values of Hep G2 cells treated with DHAA-F for 24,48 and 72 h were 56.8±4.4,40.2±3.4 and（24.2±2.4）μmol·L^（-1）,respectively. Cells treated with DHAA-F 20,40 and 80 μmol·L^（-1）for 24 h became round,deformative and shrunken. Apoptosis rates of DHAA- F groups increased to（12.3 ± 1.7）%,（28.8 ± 3.2）% and（61.8 ± 4.6）% from（6.4±0.6）% of control group（P〈0.01）. Compared with control group,phosphorylation of JNK and P38 was increased after DHAA-F treatment（P〈0.01）that down-regulated BCL-2,up-regulated BAX and activated cleaved caspase- 9/3,but the phosphorylation of the extracellular signal- regulated protein kinase（ERK1/2）was not affected. Addition of 10 μmol · L^（-1）SP600125（JNK inhibitor）and10 μmol·L^（-1）SB203580（P38 inhibitor）remarkably suppressed JNK and P38 phosphorylation induced by DHAA-F 80 μmol·L^（-1）. Moreover,SP600125 or SB203580 treatment blocked DHAA-F-can induced down-regulation of BCL-2,up-regulation of Bax and caspase-
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2016年第11期1149-1155,共7页
Chinese Journal of Pharmacology and Toxicology
基金
河南科技大学博士科研启动基金(4020/13480023)~~
关键词
脱氢枞胺对氧苯甲醛
丝裂原活化蛋白激酶
细胞凋亡
肝细胞癌
dehydroabietylamine-fluorobenzaldehyde
mitogen-activated protein kinase
apoptosis
hepatocellular carcinoma
