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

Abstract

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 (P < 0.05). BFA significantly increased the protein and mRNA levels of caspase-3 and PARP-1 (P < 0.05) compared to control and DMSO-treated cells, indicating that BFA induced apoptosis in HepG2 cells by increasing the expression of caspase-3 and PARP-1. The induction of apoptosis by BFA could be further significantly enhanced by co-incubation with TM. In addition, BFA significantly increased the mRNA levels of BiP, PERK and ATF4 (P < 0.05) compared to control and DMSO-treated cells. After co-incubation of BFA and TM, the protein levels of BiP, p-PERK, p-eIF2α and CHOP were significantly increased, indicating that TM could enhance BFA-induced ER stress in HepG2 cells through the PERK-eIF2α-ATF4-CHOP pathway.

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-eIF2α-ATF4-CHOP pathway. The findings highlight the therapeutic potential of the combined use of BFA and TM in treating HCC.

Keywords

Brefeldin A (BFA) / Tunicamycin (TM) / Hepatocellular carcinoma (HCC) / Apoptosis / Endoplasmic reticulum stress (ERS) / Caspase-3

Data availability statement

The data that support the findings of this work are available from the corresponding author upon reasonable request.

Authors’ contributions

Minghong Li and Mengyi Duan contributed equally to this work and should be considered co-first authors. Minghong Li: Writing e original draft, Validation, Software, Methodology, Investigation. Mengyi Duan: Writing e original draft, Methodology, Investigation. Ying Yang: Methodology, Investigation. Xingdao Li: Methodology, Investigation. Dan Li: Methodology, Investigation. Wenting Gao: Methodology, Investigation, Formal analysis. Xiao-tong Ji: Writing e review & editing, Formal analysis. Jianying Bai: Writing e review & editing, Validation, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptuali-zation. All authors have read and approved the final version of the manuscript.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 30972445) and the Natural Science Foundation of Shanxi Province of China (No. 202203021211231).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.livres.2025.01.004.

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