Background/Objectives: Vitex agnus-castus has been traditionally used to treat hormonal disorders, and recent evidence suggests its potential anticancer properties. However, its effects on gastric cancer remain unclear. Methods: This study examined the cytotoxic, apoptotic, and anti-metastatic effects of hydroalcoholic Vitex agnus-castus seed extract in gastric cancer cells. Antioxidant capacity (DPPH, ABTS) and total phenolic and flavonoid contents were analyzed. Cytotoxicity was assessed using the MTT assay in HGC27, MKN45, and AGS gastric cancer cell lines and CCD-1072Sk fibroblasts. Apoptosis, mitochondrial membrane potential (MMP), and cell cycle changes were evaluated via Annexin V-FITC/PI, Rhodamine 123, and PI staining, respectively. RT-qPCR and gene enrichment analyses were conducted to investigate the molecular mechanisms. Apoptosis-related protein expression was analyzed through enzyme-linked immunosorbent assay (ELISA). Results: The extract exhibited high antioxidant activity and a significant phenolic and flavonoid content. It reduced cell viability in a dose-dependent manner in gastric cancer cells, while exerting low toxicity in fibroblasts. It significantly increased apoptosis, induced G0/G1-phase cell cycle arrest, upregulated pro-apoptotic genes (CASP3, CASP7, TP53, BCL2L11), and downregulated anti-apoptotic genes (XIAP, NOL3). Gene enrichment analysis highlighted pathways like apoptosis, necrosis, and cysteine endopeptidase activity. The extract also disrupted MMP, inhibited migration and spheroid formation, suppressed EMT markers (SNAIL, SLUG, TWIST1, N-CADHERIN), and upregulated E-CADHERIN. The expression of Caspase 3 and Bax proteins increased and Bcl2 protein decreased. Conclusions: These findings suggest that Vitex agnus-castus seed extract exerts strong anticancer effects in gastric cancer cells by promoting apoptosis, reducing proliferation, and inhibiting migration. Further studies are warranted to explore its clinical relevance.

Multidrug resistance remains a major obstacle in gastric cancer therapy and is frequently associated with aggressive phenotypes. Although ABCG2 is a well-known drug efflux transporter, its functional contribution to paclitaxel (PTX) resistance and its relationship with ERK signaling in gastric cancer remain incompletely understood. In this study, PTX-resistant gastric cancer cell models were established through prolonged drug exposure. Resistant cells exhibited cross-resistance to cisplatin and 5-fluorouracil together with enhanced drug efflux activity, invasion capacity, spheroid formation, stemness-associated marker expression, and G0/G1 enrichment. ABCG2 expression was markedly increased in resistant cells. Stable knockdown of ABCG2 restored PTX sensitivity and significantly reduced drug efflux, invasion, spheroid formation, and stemness-associated phenotypes, while increasing apoptosis and altering cell cycle distribution. ABCG2 depletion was associated with reduced ERK phosphorylation and decreased expression of ERK downstream target genes. Pharmacological inhibition of ERK signaling similarly suppressed resistance-associated phenotypes and reduced ABCG2 expression. Whereas reactivation of ERK signaling by constitutively active MEK1 partially rescued the effects of ABCG2 depletion, restoring aggressive and multidrug-resistant phenotypes. Our findings indicate that ERK signaling functionally contributes to ABCG2-associated multidrug resistance and aggressive phenotypes in PTX-resistant gastric cancer cells.