Ethyl acetate fraction of Cyperus amuricus induces autophagy and apoptosis in hepatocellular carcinoma cells via modulating the AMPK-mTOR pathways

Objective: To investigate whether the ethyl acetate fraction of Cyperus amuricus ( C. amuricus ) induces autophagy-mediated apoptosis in hepatocellular carcinoma cells via the AMPK and PI3K/Akt/mTOR signaling pathways. Methods: In vitro experiments were conducted using HepG2 and Huh7 hepatocellular carcinoma cell lines. The ethyl acetate fraction of C. amuricus was profiled by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and quantitative high-performance liquid chromatography with diode-array detection. In HepG2 cells, cytotoxicity (MTT assay), apoptosis (Annexin V/PI staining, sub-G 1 analysis, and Western blot for cleaved caspase-3, Bax/Bcl-2, and cytochrome c), autophagy (acridine orange staining, LC3-II/LC3-I ratio, Beclin-1, Atg5-Atg12, Atg7, DAPK3, and autophagic flux assays with bafilomycin A1), and signaling pathways (p-AMPK and total AMPKα, and PI3K/Akt/mTOR/p70S6K by Western blot) were assessed at 100, 150 and 200 μg/mL for 3-24 h. The role of autophagy in apoptosis was further evaluated using the autophagy inhibitors 3-methyladenine and bafilomycin A1. Cytotoxicity, apoptosis, and autophagy were also assessed in Huh7 cells at 100-200 μg/mL for 24 h. Results: The ethyl acetate fraction of C. amuricus significantly reduced viability dose-dependently with an IC 50 of 150 μg/mL (HepG2) and 165 μg/mL (Huh7), activated intrinsic apoptosis, and induced time-dependent autophagy with confirmed flux ( P < 0.05). The fraction markedly enhanced AMPK phosphorylation while suppressing the PI3K/Akt/mTOR/p70S6K signaling ( P < 0.05). Cotreatment with 3-methyladenine reduced apoptosis from 30.9% to 26.0% and decreased the levels of p-AMPKα, p-mTOR, Atg7, and Beclin-1, and increased LC3-H/LC3-I ratio ( P < 0.05), while bafilomycin A1 co-treatment reduced apoptosis to 24.3% in HepG2 cells and confirmed autophagic flux as a pro-apoptotic mechanism, collectively establishing a functional role of autophagy in C. amuricus fraction-induced cell death. Conclusions: The ethyl acetate fraction of C. amuricus induces autophagy-mediated apoptosis via AMPK activation and PI3K/Akt/mTOR inhibition. Therefore, it holds great potential as a phytotherapeutic candidate for hepatocellular carcinoma, which requires further in vivo validation.