Effects of the combination of brefeldin A and tunicamycin on endoplasmic reticulum stress and apoptosis in human normal hepatocytes

Brefeldin A (BFA) and tunicamycin (TM) induce a mild, non-canonical endoplasmic reticulum (ER) stress response in normal human hepatocytes. Treatment with BFA + TM upregulates BiP and ATF4 without activating the PERK-eIF2α-CHOP axis, leading to weak, caspase-12-dependent but caspase-3-independent apoptosis. These findings highlight the need to evaluate the potential hepatotoxicity of BFA-based therapies. • Brefeldin A (BFA) and tunicamycin (TM), alone or combined, significantly reduce the viability of human normal hepatocytes (HL-7702). • BFA alone and BFA + TM induce weak apoptosis in HL-7702 cells, accompanied by increased caspase-12 expression and decreased PCNA levels. • The pro-apoptotic effect of BFA is enhanced by TM but remains independent of CHOP and caspase-3 activation. • BFA upregulates BiP and ATF4 expression at both mRNA and protein levels, indicating ER stress induction via a non-classical PERK-eIF2α-CHOP pathway. • The findings highlight the hepatotoxic potential of BFA and TM, underscoring the need for careful safety assessment in future clinical applications. Due to its ability to inhibit the growth of hepatoma cells, brefeldin A (BFA) has been considered a promising drug candidate for liver cancer. However, there is limited research on its safety profile and potential impacts when administered alone or in combination with other anticancer drugs. To evaluate the safety of BFA in combination with tunicamycin (TM, a candidate anticancer drug) in human normal liver cells (HL-7702) in terms of its ability to induce endoplasmic reticulum (ER) stress and apoptosis. HL-7702 cells were exposed to BFA (0–2.5 mg/L) and TM (0–5 mg/L), either alone or in combination, for 24 h. Cell viability was measured using the CCK-8 assay, and apoptotic rates were determined using flow cytometry. The mRNA and protein levels of key factors related to cell proliferation, ER stress, and apoptosis were determined using quantitative RT-PCR and Western blot, respectively. BFA and TM, either alone or in combination, significantly reduced the viability of HL-7702 cells. BFA alone and BFA + TM combination could weakly induce apoptosis, increase the expression of caspase 12, and reduce the protein level of proliferating cell nuclear antigen (PCNA). BFA alone and BFA + TM combination could significantly increase the mRNA and protein levels of binding immunoglobulin protein ( BiP ) and activating transcription factor 4 ( ATF4 ), but did not affect the mRNA and protein levels of C/EBP homologous protein ( CHOP ) and poly (ADP-ribose) polymerase-1 (PARP-1). This study demonstrates that BFA, alone and in combination with TM, exerts mild pro-apoptotic effects on HL-7702 cells, independent of the CHOP and caspase-3 pathways. These findings underscore the necessity of evaluating the potential hepatotoxicity of BFA-based therapies, particularly in combination treatments, to ensure their safe clinical application.