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

Abstract

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-G1 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 IC50 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.

Keywords

Cyperus amuricus / Apoptosis / Autophagic flux / Hepatocellular carcinoma / AMPK / PI3K/Akt/mTOR

Conflict of interest statement

The authors declare no competing interests.

Funding

This research is funded by Vietnam National Foundation for Science and Technology Development under grant number 108.062021.02. This research is also supported in part by the research facilities of Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam.

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon request.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work, the authors used ChatGPT (OpenAI) in order to assist with language editing. After using this tool, the authors reviewed and edited the content as needed and took full responsibility for the content of the published article.

Authors’ contributions

HHPT conceptualized the study, developed the methodology, performed experiments, conducted formal analysis, contributed to visualization, resources, and validation, and wrote the original draft and reviewed and edited the manuscript. KCT performed experiments, and contributed to methodology, formal analysis, and validation. MQP performed experiments, and contributed to methodology, formal analysis, visualization, resources, and validation. TLN conceptualized the study, developed the methodology, curated data, supervised the project, contributed to validation and resources, acquired funding, wrote the original draft, and reviewed and edited the manuscript.

Publisher’s note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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