To examine the impact of Piperlongumine (PL) on the proliferation, migration, invasion, cell cycle progression, and apoptosis in esophageal squamous cell carcinoma (ESCC) cells, as well as to elucidate the underlying molecular mechanisms. The suppressive effects of PL on the viability of ESCC cells were assessed using the CCK-8 assay, bright field imaging, and colony formation assays. Apoptosis induction and cell cycle disruption by PL were evaluated using flow cytometry. The impact of PL on ESCC cell migration and invasion was examined through scratch healing and Transwell assays. Differential gene expression analysis of ESCC tumor and normal tissues from the GSE29886 dataset, integrated with network pharmacology predictions, was conducted to identify core genes and molecular mechanisms involved in PL action. Key protein expression levels in the apoptosis, epithelial-mesenchymal transition (EMT), and PI3K/AKT signaling pathways were quantified by Western blotting. The CCK-8 and colony formation assays demonstrated that PL effectively suppressed cell viability and proliferation in ESCC. Flow cytometry revealed that PL down-regulated CDK1 expression, resulting in G2/M phase arrest, and promoted apoptosis by decreasing Bcl-2 levels and increasing cleaved caspase-3 and PARP. The scratch and Transwell assays indicated that PL inhibited ESCC cell migration and invasion, down-regulated the EMT-associated proteins Vimentin and N-cadherin, and up-regulated E-cadherin. Western blotting confirmed the down-regulation of P-PI3K and P-AKT, indicating the inhibition of the PI3K/AKT pathway by PL. These findings offer a pharmacological foundation for the development of PL as a potential phytotherapeutic agent for the clinical management of ESCC.
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