Exploring Nonactin’s Anticancer Activity against A549 lung cancer cells: In Vitro Cytotoxicity, Gene Expression, and In Silico Docking and MD Simulation

Lung cancer remains the leading cause of cancer-related mortality worldwide, highlighting the urgent need for effective therapeutic strategies. Drug repurposing has emerged as a promising approach for identifying new anticancer applications of existing compounds. This study investigated the anticancer potential and molecular mechanism of Nonactin, a macrotetrolide ionophoric antibiotic, against A549 lung cancer cells using integrated in vitro and Computational approaches. Nonactin exhibited potent dose-dependent cytotoxicity with an IC₅₀ of 4.17 µg/mL 48 h of treatment. Hoechst-33342 and FITC-Annexin V/PI staining confirmed apoptotic cell death, while no significant intracellular ROS generation was observed, indicating a ROS-independent mechanism. qPCR analysis demonstrated significant upregulation of TP53, TNF-α, FAS, CASP9, CYCS , and BID , whereas PARP1, NF-κB, CASP8 , and BAX were downregulated, suggesting activation of the p53-mediated intrinsic mitochondrial apoptotic pathway with limited involvement of the extrinsic pathway. Molecular docking revealed strong binding affinities of nonactin toward p53 (-11.7 kcal/mol), NF-κB (-9.7 kcal/mol), and TNF-α (-9.5 kcal/mol). MD simulations and MM-PBSA analyses confirmed stable protein-ligand interactions, while Gene-TF, Gene-miRNA, PPI, and lung tissue-specific network analyses further supported apoptosis-associated regulatory hubs & inflammatory signaling pathways. Collectively, these findings identify Nonactin as a promising drug repurposing candidate for lung cancer therapy and provide a foundation for future in vivo validation and translational research.