Targeting PI3K/AKT signaling in NSCLC: Dual in silico and in vitro validation of Withanolide B as a potent AKT modulator.

[ETHNOPHARMACOLOGICAL RELEVANCE] Withania somnifera (L.) Dunal (Ashwagandha) has long been utilized in traditional medicine for its rejuvenating and anticancer properties. Withanolides, its major bioactive constituents, are known to influence multiple oncogenic pathways. Elucidating the molecular mechanisms underlying these effects is essential for translational development.

[AIM OF THE STUDY] To evaluate the potential of Withanolide B (WB) as a multi-isoform AKT-modulating candidate targeting PI3K/AKT-associated survival signaling in non-small cell lung carcinoma (NSCLC) using integrated in silico and in vitro approaches.

[MATERIALS AND METHODS] Molecular docking and 100-ns molecular dynamics (MD) simulations were conducted to examine WB interactions with AKT1, AKT2, and AKT3 isoforms. Binding free energies were estimated using MM-PBSA analysis. Drug-likeness and toxicity were predicted using SwissADME and DataWarrior. Experimental validation was performed in A549 NSCLC cells and L132 normal lung epithelial cells using MTT assay, ROS quantification, mitochondrial membrane potential (ΔΨm) analysis, apoptosis staining, and cell cycle profiling.

[RESULTS] WB demonstrated favorable binding affinity toward all AKT isoforms with stable MD trajectories and energetically favorable MM-PBSA profiles, particularly for AKT2. In vitro, WB significantly reduced A549 cell viability (IC = 5 μM), induced ROS accumulation, mitochondrial depolarization, chromatin condensation, apoptosis, and S-phase cell cycle arrest. Importantly, WB exhibited reduced cytotoxicity toward L132 normal lung epithelial cells, indicating cancer-selective activity.

[CONCLUSIONS] Withanolide B exhibits multi-level anticancer activity in NSCLC, potentially involving modulation of AKT-associated survival signaling. While direct biochemical validation of AKT phosphorylation was not performed, integrated computational and functional evidence supports its therapeutic promise. These findings provide mechanistic insight into the traditional use of W. somnifera and warrant further preclinical investigation.