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

Objective 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 and 48 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 and 48 h resulted in death of HepG2 cells in a dose-dependent manner. Cell viability of 0.1-5 μg/mL 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/mL BFA significantly increased the gene expression of COPⅠ, Arf1, COPⅡ and, Sar1b (F of 24 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 and 170 respectively, P < 0.05). After treatment with 2.5 μg/mL 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 and 48 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/mL 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/mL 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.