Ligustilide Promotes Gastric Cancer Cell Apoptosis via Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction and Autophagy

Ligustilide is a natural benzoquinone derivative with many pharmacological properties, including anticancer and anti-inflammatory activities.Here, we investigated the antitumor efficacy of ligustilide in gastric cancer.MKN74 and AGS cells were treated with 20, 60, and 100 M ligustilide.The antitumor efficacy of ligustilide was studied with MTT assays, colony formation assays, and flow cytometry.Apoptosis-, endoplasmic reticulum stress-, and autophagy-associated proteins were examined by A c c e p t e d M a n u s c r i p t immunoblotting and immunofluorescence.JC-1 staining was used to detect mitochondrial membrane potential in cancer cells.Tumor-bearing mice were intraperitoneally injected with 5 mg/kg ligustilide.Tumor weight and tumor volume were measured after 20 days of administration.We demonstrated that ligustilide suppressed gastric cancer cell proliferation by inducing cell cycle arrest and apoptosis.Mechanistically, ligustilide disrupted mitochondrial function by increasing the Bax/Bcl-2 ratio, triggering cytochrome c release into the cytoplasm, and activating caspase-3 and PARP cleavage, ultimately leading to mitochondrial membrane potential loss.Furthermore, ligustilide enhanced autophagy, as evidenced by elevated LC3-II/LC3-I ratio and ATG5 expression, accompanied by reduced p62 levels.This autophagic response was mediated through endoplasmic reticulum stress via the PERK-eIF2-CHOP pathway.Notably, the proapoptotic effects of ligustilide were attenuated by 4-phenylbutyric acid (an ER stress inhibitor) or 3-methyladenine (an autophagy inhibitor).Consistent with in vitro findings, 4-phenylbutyric acid also abrogated the antitumor efficacy of ligustilide in vivo by concurrently suppressing apoptosis and autophagy.Overall, ligustilide triggers mitochondrial dysfunction, autophagy, and endoplasmic reticulum stress, collectively promoting gastric cancer cell death and suppressing survival.