Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway.

Background and aims: Hepatocellular carcinoma (HCC) is a malignant tumor with a high mortality rate, but there are still no effective treatments. The aim of this study was to investigate the anticancer potential of the combined use of brefeldin A (BFA) and tunicamycin (TM) in HepG2 cells, as well as the underlying mechanisms. Methods: HepG2 cells were treated with different concentrations of BFA (0.1-2.5 mg/L) and TM (1-5 mg/L) for 24 h. DMSO (0.1 %, v/v) was used as a vehicle control. Cell viability and cell migration were measured using MTT assay and scratch wound assay, respectively. Apoptosis was detected using flow cytometry and acridine orange (AO) staining. The protein and mRNA levels of various factors involved in apoptosis (poly (ADP-ribose) polymerase-1 (PARP-1), caspase-12, caspase-3, and stearoyl-CoA desaturase 1) and endoplasmic reticulum (ER) stress (binding immunoglobulin protein (BiP), protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, phosphorylation of eukaryotic translation initiation factor 2alpha (p-eIF2α), activating transcription factor (ATF) 4, and C/EBP homologous protein (CHOP)) were measured using Western blotting and qRT-PCR, respectively. Results: Both BFA and TM alone significantly reduced the viability of HepG2 cells in a dose-dependent way. The co-incubation with TM (1 mg/L) further significantly reduced the viability of HepG2 cells treated with BFA (0.25 mg/L) alone ( Conclusions: BFA could induce apoptosis and ER stress, and TM could enhance the ability of BFA to induce apoptosis and ER stress in HepG2 cells through the PERK-eIF2ɑ-ATF4-CHOP pathway. The findings highlight the therapeutic potential of the combined use of BFA and TM in treating HCC.