Mechanistic Studies of Anticancer Effects of Salicylaldehyde Benzoylhydrazone in 2D/3D Lung Cancer Cell Cultures.

[BACKGROUND/AIM] 3D culture models have become essential in cancer research, as they reflect tumor biology more accurately and predict responses more reliably. Numerous studies have proven that cells cultured in 3D systems exhibit greater resistance to anticancer agents than those grown in 2D monolayers. In this study, the anticancer activity and underlying mechanisms of salicylaldehyde benzoylhydrazone (SBH) were investigated in A549 lung cancer cells cultured under 2D and 3D spheroid (SP) conditions.

[MATERIALS AND METHODS] Cell viability was assessed using the MTT and ATPlite 3D assays. Apoptosis and cell cycle distribution were analyzed with a Muse Cell Analyzer. Mitochondrial membrane potential and the activities of caspase-9 and caspase-3/7 were determined using the JC-10 assay kit, Caspase-Glo 9, and Caspase-Glo 3/7 assays, respectively. Cell migration and invasion were examined by Transwell assays. Effects on cell survival, motility, cell cycle regulators, and apoptotic signaling were elucidated by immunoblotting. Molecular docking and molecular dynamics simulations were conducted to model and evaluate the interactions of SBH with JAK2, STAT3, and FAK.

[RESULTS] Dissociated SP-culture cells displayed increased responsiveness to SBH treatment compared with corresponding 2D cultures. SBH primarily exerted cytostatic effects in both 2D and SP cells and reduced their migratory and invasive capabilities. Mechanistic analyses revealed that SBH treatment significantly decreased the phosphorylation levels of key oncogenic kinases, including JAK2, STAT3, and FAK. Molecular docking analysis and molecular dynamics simulations further supported these findings by confirming the interactions of SBH with JAK2 and FAK.

[CONCLUSION] SBH may mediate the cytostatic and anti-metastatic effects by targeting the JAK2/STAT3 and FAK/MYLK signaling axes.