Gallic acid-conjugated 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone induces apoptosis and downregulates PI3K/Akt signaling through VEGFR-2 targeting in non-small cell lung cancer (NSCLC).

Lung cancer is the leading cause of cancer-related mortality worldwide. Phytochemical compounds, particularly chalcones derived from Syzygium nervosum A. Cunn. ex DC., have demonstrated notable anticancer potential. The major constituent, 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC, compound 1), exhibits anticancer effects against multiple cancer types. Chemical modifications of DMC, including conjugation with gallic acid (compound 2), have been explored to enhance its pharmacological activities. This study evaluated the gallic acid-2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) hybrid derivative (compound 3) using an integrated experimental and computational workflow. In vitro 2D cytotoxicity assays demonstrated potent antiproliferative activity, with IC₅₀ values of 3.93 ± 0.47 μM in A549 and 4.88 ± 1.42 μM in non-small cell lung cancer (NSCLC) cells, while showing reduced cytotoxicity in normal fibroblasts (MRC-5; IC₅₀ = 10.48 ± 1.83 μM) compared with osimertinib. Flow cytometry confirmed a three-fold increase in apoptosis relative to controls. Pharmacological pathway analysis identified VEGFR-2 as a central regulatory node in PI3K/Akt signaling. The cytotoxic potential of compound 3 was further validated in 3D spheroid models using confocal imaging and LDH release assays. mRNA expression analysis confirmed this signaling pathway, revealing significant downregulation of PTGER4, AKT1, PDPK1, and PRKCA. Molecular docking and in silico conformational analyses demonstrated strong binding affinity of compound 3 to the VEGFR-2 kinase domain, stabilizing critical active-site residues and supporting a structure-based mechanism of action. Collectively, these findings validate VEGFR-2 as a therapeutic target and highlight compound 3 as a promising scaffold for structure-based drug development against non-small cell lung cancer.