Hispidulin-mediated inhibition of lung cancer progression through PI3K/AKT Signaling: A combined bioinformatic analysis and experimental investigation.

[UNLABELLED] Non-small cell lung cancer (NSCLC) continues to be one of the foremost causes of cancer-associated mortality globally, underscoring the critical need for novel therapeutic agents with broad, multi-targeted efficacy. In the present study, the anticancer potential of hispidulin, a naturally occurring prenylated flavonoid, was evaluated against NSCLC using an integrated strategy that combined network pharmacology, molecular docking, and in vitro validation. Network pharmacology analysis identified PI3K, AKT1, mTOR, and BCL2 as key hub targets, all of which play pivotal roles in regulating cell survival, proliferation, and resistance to apoptosis. Molecular docking studies revealed strong binding interactions between hispidulin and these core proteins, indicating its potential to directly modulate their activity. In vitro assays demonstrated dose- and time-dependent cytotoxic effects, with IC₅₀ values of 129.63 µM at 24 h and 88.45 µM at 48 h. Morphological assessment by phase-contrast microscopy showed cellular shrinkage, while propidium iodide staining confirmed nuclear condensation and fragmentation. Elevated intracellular ROS levels detected by DCF-DA staining suggested the involvement of oxidative stress-mediated apoptotic pathways. Flow cytometric analysis further revealed G0/G1-phase cell cycle arrest and a significant increase in apoptotic cell populations following prolonged exposure to hispidulin. Consistent with these findings, quantitative RT-PCR analysis demonstrated significant downregulation of PI3K, AKT1, mTOR, and BCL2 transcripts, validating the molecular mechanism underlying hispidulin-induced cytotoxicity. Collectively, these results highlight hispidulin as a promising natural anticancer agent against NSCLC, exerting its effects through multi-targeted suppression of the PI3K/AKT/mTOR signaling axis and induction of apoptosis via ROS-mediated mechanisms.

[SUPPLEMENTARY INFORMATION] The online version contains supplementary material available at 10.1007/s10616-026-00958-0.