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 Cu 2+ ). Cell viability was assessed using the MTT assay, and morphological changes were observed under a microscope. Cell proliferation and m