布雷菲尔德菌素A经内质网应激途径诱导的肝癌细胞HepG2发生p53依赖性死亡

Brefeldin A-induced p53-dependent Death in Hepatocellular Carcinoma Cells HepG2 via Endoplasmic Reticulum Stress Pathway

目的探讨布雷菲尔德菌素A(BFA)作为肝脏肿瘤治疗药物的作用机制和可能的临床应用价值。方法采用细胞毒性试验(MTT法)观察BFA对肝癌细胞Hep G2的细胞毒性作用,采用定量荧光PCR法、蛋白杂交法观察BFA对Hep G2细胞有关囊泡转运相关、内质网应激相关基因和蛋白表达的影响。结果用不同浓度的BFA处理肝癌细胞24 h和48 h后,可引起肝癌细胞死亡,并呈现剂量依赖关系。(0.1~5)μg/m L BFA处理组细胞存活率与阴性对照组比较,差异具有统计学意义(24 h和48 h的H值分别为45.7和45.9,P<0.01)。2.5μg/m L BFA可引起HepG2细胞内COPI、Arf1、COPII和Sar1b的基因表达显著升高(24 h实验组的F值分别为72.5、41.4、107和134,P<0.05;48 h实验组的F值分别为69.2、51.4、49.8和170,P<0.05)。2.5μg/m L BFA处理24 h、48 h后可引起HepG2细胞内BiP(GRP78)和calnexin基因表达水平明显升高,其中BiP(GRP78)在24 h和48 h的基因表达分别为阴性对照组的46倍和36倍,calnexin在24 h和48 h的基因表达分别为对照组的10倍和5倍。2.5μg/m L BFA处理24 h、48 h后可引起HepG2细胞内Bi P(GRP78)蛋白表达明显升高,约为对照组的2倍,calnexin的表达明显减少,几乎无法检测到。2.5μg/m L BFA引起HepG2细胞内p-AMPKα(Thr 172)和p-p53蛋白表达明显增加。结论BFA可通过内质网应激途径引起肝癌细胞发生p53依赖的死亡,对肝癌细胞有一定的杀伤作用。

Objectives To explore the mechanism and possible clinical value of Brefeldin A( BFA) as a therapeutic drug for liver tumors. Methods After HepG2 cells exposed to different concentrations of BFA for 24 h and48 h,MTT assay was used to test their cytotoxicity to HepG2 cells. qRT-PCR was used to study the gene expression level and western blotting was used to detect the protein expression of vesicles transport and endoplasmic reticulum( ER) stress related factors. Results Treatment of hepatocytes with different concentrations of BFA for 24 h and48 h resulted in death of HepG2 cells in a dose-dependent manner. Cell viability of 0. 1-5 μg/m L BFA group was significantly decreased compared with negative control( H were 45. 7 and 45. 9 in 24 h and 48 h,respectively,P<0. 01). 2. 5 μg/m L BFA significantly increased the gene expression of COPI,Arf1,COPII and,Sar1 b( F of24 h groups were 72. 5,41. 4,107 and 134 respectively,P<0. 05;F of 48 h groups were 69. 2,51. 4,49. 8 and170 respectively,P < 0. 05). After treatment with 2. 5 μg/m L BFA for 24 h and 48 h,the expression of BiP( GRP78) and calnexin in HepG2 cells were significantly increased. The expressions of BiP( GRP78) at 24 h and48 h were 46-fold and 36-fold,respectively. The gene expression of calnexin at 24 h and 48 h was about 10 times and 5 times that of the control group,respectively. After treatment with 2. 5 μg/m L BFA for 24 h and 48 h,the expression of BiP( GRP78) protein in HepG2 cells was significantly increased,which was about 2 times that of the control. The expression of calnexin was significantly reduced and almost impossible to be detected. 2. 5 μg/m L BFA significantly increased the protein expressions of p-p53 and pAMPKα( Thr 172). Conclusions Brefeldin A treatment is capable to induce cell death to HepG2 by p-p53 dependent ER stress pathway.