Work

Ailanthone targets mitophagy-loaded SIRT4 to facilitate the deacetylation of HIF1α, triggering oxidative stress for gastric carcinoma treatment

Year: 2025

Type: article

Abstract: Background: Gastric carcinoma holds a great threat to human health globally and needs to be solved urgently. Ailanthone (AIL) shows anti-tumor characteristics in different kinds of cancers, including gastric cancer, however, its deep anti-gastric cancer mechanisms still remain to be uncovered.Purpose: The goal of this research was to explore the efficacy and underlying molecular mechanisms of AIL in treating gastric carcinoma.Methods: The anti-proliferative effects of AIL on gastric cancer cells were evaluated through MTT, colony formation and EdU incorporation assays. Transcriptomics analysis was conducted to explore the potential targets and signaling pathways affected by AIL. Mechanistic experiments, including western blot, flow cytometry, single cell gel electrophoresis (SCGE), glutathione assay, measurement of the MDA content, immunofluorescence staining, and siRNA transfection were employed to reveal the regulatory mechanisms of AIL on reactive oxygen species (ROS) production and mitochondrial dysfunction-mediated cell apoptosis. Moreover, transmission electron microscopy, mitochondria isolation, and co-immunoprecipitation assay (Co-IP) were applied to confirm the inhibitory effect of AIL on mitophagy. Finally, the xenograft mice bearing MGC-803 cells and HE staining experiment were conducted to observe AIL's effect on tumor growth and its toxicity.Results: AIL significantly suppressed gastric cancer cell proliferation, affected oxidative stress pathway, caused ROS accumulation and DNA damage, verified by a long tail detected through SCGE and elevated γ-H2AX protein levels. Moreover, AIL induced mitochondrial dysfunction-mediated cell apoptosis, triggered by ROS accumulation. By detecting autophagy-related proteins, we found that AIL inhibited p62 and LC3 protein levels and activated the mTOR signaling pathway. Importantly, rapamycin attenuated AIL-induced ROS production and apoptosis, suggesting the protective role of autophagy against ROS. By using transmission electron microscope, we further confirmed that the autophagy inhibited by AIL was mitophagy which was also validated by decreased MFN2, NIX and PARKIN levels. As a result, AIL accumulated the mitophagy substrate deacetylase SIRT4, thus resulting in the deacetylation and subsequent loss of the transcription factor HIF1α stability. Moreover, specific knockdown of SIRT4 markedly restored the acetylation level of HIF1α repressed by AIL. This reduction of HIF1α stability ultimately resulted in the decreased expression of the antioxidant protein HO-1. Finally, AIL inhibited the tumor growth in xenograft mice bearing MGC-803 cells with low toxicity.Conclusions: Our results unveiled that AIL could target mitophagy-loaded SIRT4 to regulate redox homeostasis, which ultimately induced cell apoptosis and inhibited gastric cancer growth. Our research provided powerful evidence for the application of AIL in gastric cancer treatment.

Source: Phytomedicine

DOI: 10.1016/j.phymed.2025.157283