Piperine-induced AMPK activation and cell cycle arrest: insights from molecular simulations and experimental validation in breast cancer.

Abstract

<p>Breast cancer remains a leading cause of mortality among women, underscoring the need for novel, low-toxicity therapeutic strategies. Piperine, a bioactive alkaloid derived from Piper nigrum, has shown promising anticancer activity; however, its mechanistic link to AMPK activation remains insufficiently explored. In this study, we investigated the role of AMPK modulation in piperine-induced apoptosis in MCF-7 (luminal A, ER-positive) breast cancer cells by integrating in silico molecular docking and 100&nbsp;ns molecular dynamics (MD) simulations with comprehensive in vitro validation. Molecular docking revealed that piperine binds at the AMPK α-β interface (ADaM-like site) with a favorable binding affinity (- 6.3&nbsp;kcal/mol), comparable to the reference AMPK activator metformin (- 6.8&nbsp;kcal/mol). MD simulations demonstrated stable ligand-protein interactions and conformational stabilization of AMPK over 100&nbsp;ns. Functionally, piperine exhibited a time-dependent cytotoxic effect with IC₅₀ values of 81&nbsp;µM (24&nbsp;h), 44&nbsp;µM (48&nbsp;h), and 15&nbsp;µM (72&nbsp;h) as determined by MTT assay. Annexin V-FITC/PI analysis showed significant apoptosis induction, with total apoptotic populations of 37.6 ± 2.95% at 50&nbsp;µM and 69.3 ± 2.85% at 100&nbsp;µM. Cell cycle analysis revealed a marked arrest at the G2/M phase (~ 20-22%).Additionally, piperine significantly inhibited cancer cell migration, reducing wound closure at 24&nbsp;h to 29.5 ± 0.3% (50&nbsp;µM) and 18.8 ± 0.2% (100&nbsp;µM) compared to 45.3 ± 0.4% in controls, and at 48&nbsp;h to 48.1 ± 0.2% and 27.6 ± 0.3%, respectively (control: 72.4 ± 0.6%). Collectively, these findings demonstrate that piperine effectively modulates AMPK signaling, correlating with apoptosis induction, cell-cycle arrest, and impaired migratory capacity in breast cancer cells, thereby highlighting its potential as an AMPK-targeting therapeutic candidate for luminal A breast cancer.</p>