Birgül, KaanAtlıhan, İremDere, DamlaYelekçi, KemalTiber, Pınar MegaOrun, OyaKüçükgüzel, Sükriye Güniz2025-09-102025-09-1020250045-20681090-212010.1016/j.bioorg.2025.1088502-s2.0-105012445274https://doi.org/10.1016/j.bioorg.2025.108850https://hdl.handle.net/20.500.14627/1172This study focused on the synthesis of novel nimesulide semicarbazone derivatives and the evaluation of their cytotoxic potential against luminal-A (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell lines. Additionally, their effects on mitochondrial membrane potential (MMP), apoptosis, and mitogen-activated protein kinase (MAPK) pathway modulation were investigated. Breast cancer remains the most prevalent malignancy among women, with luminal-A and triple-negative subtypes posing significant therapeutic challenges due to drug resistance and the lack of effective targeted treatments. The MAPK pathway plays a crucial role in breast cancer progression, making its inhibition a promising therapeutic approach. Non-steroidal anti-inflammatory drugs (NSAIDs), particularly nimesulide, have demonstrated anticancer potential beyond their well-established anti-inflammatory properties. Accordingly, the semicarbazone moiety was incorporated into the molecular scaffold to enhance the antiproliferative efficacy of nimesulide derivatives, as it has been reported to exhibit cytotoxic and apoptosis-inducing effects across various cancer cell lines. A series of nimesulide semicarbazone derivatives (5a–m) were synthesized through multi-step reactions and characterized using elemental analysis, FT-IR, 1H NMR, 13C NMR and Mass spectroscopy (5e). In silico studies were performed to predict their binding affinities to MAPK12. The cytotoxic effects of the synthesized compounds were assessed by determining IC<inf>50</inf> values in MCF-7 and MDA-MB-231 cell lines (CCK8 test). Compounds exhibiting strong cytotoxic activity were further examined for their impact on MMP depolarization (JC-1 assay), apoptosis induction (Annexin V-FITC/PI staining), and MAPK pathway modulation (Western blotting of p-ERK and ERK protein). Molecular docking results indicated that the synthesized compounds exhibited favorable interactions with MAPK12, with compound 5e showing one of the highest binding affinity (−9.29 kcal/mol, Ki = 0.154 μM). Cytotoxicity assays revealed that compound 5e had the lowest IC<inf>50</inf> values (11.77 ± 0.26 μM in MCF-7; 20.72 ± 0.25 μM in MDA-MB-231), demonstrating significantly higher cytotoxicity than nimesulide. JC-1 assays confirmed that compound 5e induced MMP depolarization at higher concentrations, suggesting apoptosis activation. Flow cytometry analysis further validated a substantial increase in apoptotic cell populations following treatment with compound 5e. Western blot results showed a dose-dependent decrease in p-ERK levels in both MCF-7 and MDA-MB-231 cells, confirming MAPK pathway inhibition. These findings support that nimesulide-based semicarbazones, particularly compound 5e, exhibit potent antiproliferative and pro-apoptotic activity via MAPK pathway modulation, offering a promising avenue for the development of targeted breast cancer therapies. © 2025 Elsevier B.V., All rights reserved.eninfo:eu-repo/semantics/closedAccessApoptosisBreast CancerMAPKNimesulideSemicarbazoneLipocortin 5Mitogen Activated Protein KinaseNimesulide2 (2 Chloro 6 Fluorobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (2,4,6 Trimethylbenzylidene) N [4 (Methylsulfonamido) 3 Henoxyphenyl]Hydrazine 1 Carboxamide2 (2,6 Dichlorobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (2,6 Difluorobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (4 Bromobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (4 Chlorobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (4 Fluorobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (4 Hydroxy 3 Methoxybenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (4 Methylbenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (4 Nitrobenzylidene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 (Pyridine 4 Ylmethylene) N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 Benzylidene N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 Carboxamide2 [4 (Trifluoromethyl)Benzylidene] N [4 (Methylsulfonamido) 3 Phenoxyphenyl]Hydrazine 1 CarboxamideLipocortin 5Mitogen Activated Protein KinaseMolecular ScaffoldNimesulideSemicarbazone DerivativeUnclassified DrugAntineoplastic ActivityApoptosisArticleBinding AffinityBreast CancerBreast Cancer Cell LineCancer GrowthCancer InhibitionCarbon Nuclear Magnetic ResonanceCell PopulationComputer ModelCytotoxicityCytotoxicity AssayDepolarizationElemental AnalysisFlow CytometryFourier Transform Infrared SpectroscopyHumanHuman CellIC50MCF-7 Cell LineMDA-MB-231 Cell LineMitochondrial Membrane PotentialMolecular DockingNecrosis RateProapoptotic ActivityProton Nuclear Magnetic ResonanceWestern BlottingArticleQ1Q116440780124