Li Minghong, Duan Mengyi, Yang Ying, Li Xingdao, Li Dan, Gao Wenting, Ji Xiaotong, Bai Jianying
Department of Environmental Health, School of Public Health, Shanxi Medical University, Taiyuan, Shanxi, China.
Department of Occupational Disease Control, Ansteel Group General Hospital, Anshan, Liaoning, China.
Liver Res. 2025 Jan 20;9(1):49-56. doi: 10.1016/j.livres.2025.01.004. eCollection 2025 Mar.
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.
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.
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 ( < 0.05). BFA significantly increased the protein and mRNA levels of caspase-3 and PARP-1 ( < 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 and ( < 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.
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.
肝细胞癌（HCC）是一种死亡率很高的恶性肿瘤，但目前仍没有有效的治疗方法。本研究旨在探讨布雷菲德菌素A（BFA）和衣霉素（TM）联合使用对HepG2细胞的抗癌潜力及其潜在机制。
用不同浓度的BFA（0.1 - 2.5mg/L）和TM（1 - 5mg/L）处理HepG2细胞24小时。使用二甲基亚砜（DMSO，0.1%，v/v）作为溶剂对照。分别采用MTT法和划痕试验检测细胞活力和细胞迁移能力。使用流式细胞术和吖啶橙（AO）染色检测细胞凋亡。分别采用蛋白质印迹法和qRT-PCR检测凋亡相关因子（聚（ADP - 核糖）聚合酶 - 1（PARP - 1）、半胱天冬酶 - 12、半胱天冬酶 - 3和硬脂酰辅酶A去饱和酶1）和内质网（ER）应激相关因子（结合免疫球蛋白蛋白（BiP）、蛋白激酶R样内质网激酶（PERK）、p - PERK、真核翻译起始因子2α的磷酸化（p - eIF2α）、激活转录因子（ATF）4和C/EBP同源蛋白（CHOP））的蛋白质和mRNA水平。
单独使用BFA和TM均能以剂量依赖的方式显著降低HepG2细胞的活力。与单独用BFA（0.25mg/L）处理的HepG2细胞共同孵育TM（1mg/L）后，细胞活力进一步显著降低（P < 0.05）。与对照和DMSO处理的细胞相比，BFA显著增加了半胱天冬酶 - 3和PARP - 1的蛋白质和mRNA水平（P < 0.05），表明BFA通过增加半胱天冬酶 - 3和PARP - 1的表达诱导HepG2细胞凋亡。与TM共同孵育可进一步显著增强BFA诱导的细胞凋亡。此外，与对照和DMSO处理的细胞相比，BFA显著增加了相关因子的mRNA水平（P < 0.05）。BFA和TM共同孵育后，BiP、p - PERK、p - eIF2α和CHOP的蛋白质水平显著升高，表明TM可通过PERK - eIF2α - ATF4 - CHOP途径增强BFA诱导的HepG2细胞内质网应激。
BFA可诱导细胞凋亡和内质网应激，TM可通过PERK - eIF2α - ATF4 - CHOP途径增强BFA诱导HepG2细胞凋亡和内质网应激的能力。这些发现突出了BFA和TM联合使用在治疗HCC方面的治疗潜力。
