Punicalagin Targets FDX1 to Induce Cuproptosis for the Treatment of Gastric Cancer

Abstract

Gastric cancer represents the fifth most common malignancy globally and the third leading cause of cancer-related deaths. Due to its insidious early symptoms and frequent metastasis at diagnosis, the survival rate remains dismal. There is thus an urgent clinical need for novel therapeutic agents. Innovative strategies combining traditional chemotherapy with interventions that induce novel cell death pathways represent a promising translational direction for improving patient outcomes. Punicalagin (PUN), a natural polyphenol derived from pomegranate, exhibits potent antioxidant and broad-spectrum antitumor activities, yet its role in gastric cancer remains understudied. Three gastric cancer cell lines (AGS, HGC27, MFC) and one normal gastric mucosal epithelial cell line (GES-1) were initially selected for in vitro experiments. The effects of PUN on gastric cancer cells and normal gastric mucosal epithelial cells were assessed through MTT assay, propidium iodide (PI) staining, and cell colony formation assays, while cell migration ability was evaluated using a scratch wound healing assay. The inhibitory effect of PUN on gastric cancer was tested in a subcutaneous tumor model in nude mice, with pathological changes in vital tissues and organs observed via hematoxylin and eosin (H&E) staining. Subsequently, transcriptome sequencing was performed, and JC-1, H2DCFDA, and DHE staining methods were employed to measure mitochondrial function and reactive oxygen species (ROS) levels in PUN-treated cells. Western blotting was used to detect the expression of apoptosis- and cell cycle-related proteins. Next, two gastric cancer cell lines (AGS, HGC27) were selected for in vitro experiments to assess the combined effects of PUN and the copper ionophore elesclomol (ES) (hereafter referred to as ES-Cu, representing the combination of ES and Cu²⁺). Cell viability was assessed using the MTT assay, and morphological changes were observed under a microscope. Cell proliferation and migration abilities were assessed via colony formation and scratch wound healing assays, respectively. Fluorescence staining was used to examine mitochondrial function and ROS levels in cells co-treated with PUN and ES-Cu. Laser confocal microscopy and Western blotting were employed to determine the oligomerization level of DLAT protein by quantifying soluble and insoluble protein expression, along with the expression of ACO2, ETFDH, FDX1, and LIAS proteins. Molecular docking, molecular dynamics simulations, immunofluorescence staining, and transfection techniques were utilized to confirm the critical role of FDX1 in copper-induced cell death following co-treatment with PUN and ES-Cu. We found that PUN could suppress the viability, proliferation, and migration of gastric cancer cells in a concentration- and time-dependent manner. Subsequent in vivo experiments demonstrated that PUN inhibited tumor growth in nude mice at a safe dosage. Besides, transcriptome sequencing and Western blot analysis revealed that PUN induced cell cycle arrest and apoptosis. Secondly, the cell death mechanism triggered by PUN was closely associated with mitochondrial stress. PUN increased intracellular ROS levels and reduced mitochondrial membrane potential. Transcriptome sequencing and proteomic analysis further revealed molecular changes at both the mRNA and protein levels, with differential gene analysis identifying potential targets and pathways. Moreover, when PUN was combined with ES-Cu, cell viability, proliferation, and migration were all suppressed, while exacerbating mitochondrial dysfunction and elevated oxidative stress. Laser confocal microscopy and Western blotting were used to assess the expression of soluble and insoluble proteins, confirming the oligomerization of the DLAT protein. Western blot also showed that PUN could regulate the expression levels of ACO2, ETFDH, FDX1, and LIAS proteins. Molecular docking, molecular dynamics simulations, and siRNA transfection were performed to confirm the critical role of ferredoxin 1 (FDX1) in copper-induced cell death. Furthermore, when used in combination with chemotherapy drugs, PUN exhibited synergistic inhibitory effects on gastric cancer growth. PUN demonstrates significant antitumor activity both in vivo and in vitro by inducing mitochondrial dysfunction, which subsequently triggers apoptosis. In combination therapy strategies, PUN can work synergistically with chemotherapy drugs to suppress gastric cancer growth. Furthermore, PUN enhances its inhibitory effect on gastric cancer by working synergistically with cuproptosis through targeting FDX1.