Abstract
Esophageal cancer remains a significant global health challenge with increasing rates of incidence and mortality, particularly esophageal squamous cell carcinoma (ESCC), with higher prevalence in Asia. This study aims to investigate the cytotoxicity and potential cell death mechanisms (i.e., apoptosis, necroptosis, ferroptosis, and pyroptosis) induced by three newly synthesized benzamidine derivatives (NBDs) against the ESCC KYSE-150 cell line. MTT cell viability assay was used to assess the cytotoxic effect of the NBDs on the ESCC cell line, KYSE-150, and the normal esophageal epithelial cell line, HET-1A. In silico molecular simulation and binding free energy calculation methods were used to validate the potential cell death targets (s) involved in ESCC cells upon interaction with the NBDs. The results showed that the cytotoxic effect of all three NBDs on KYSE-150 outperformed 5-fluorouracil (5-FU), with NBD 3 producing the highest cytotoxicity (IC50: 21.57 µM), followed by NBD 2 (IC50: 34.17 µM) and NBD 1 (IC50: 37.75 µM). Among the three NBDs, NBD 3 was the most potent in inducing cytotoxicity in KYSE-150 cells. In silico molecular docking revealed that NBD 2 and NBD 3 exhibited relatively strong binding affinities (< -7.0 kcal/mol) toward multiple protein targets, including Caspase-9, BIM BH3 peptide, BCL2 Antagonist/Killer (BAK), Tumor Necrosis Factor Receptor (TNFR), Fas Cell Surface Death Receptor (FAS), BCL2-Like 1, long isoform (BCL-XL), BCL2-Like 2 (BCL-W), BCL2-Like 10 (BCL2L10), Heat Shock Protein 70 (Hsp70), Heat Shock Protein 90 (Hsp90), Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1), Dynamin-1-Like Protein (DNM1L), Apoptosis-Inducing Factor, Mitochondria-Associated 2 (AIFM2), and Absent in Melanoma 2 (AIM2), molecular simulation results further confirmed the stability, structural compactness and residual flexibility. In addition, the binding free-energy calculations characterized the relative binding strengths of these derivatives toward the selected pathway targets. These computational results, together with the docking-derived interaction profiles, support the prediction that these programmed cell-death pathways may be potentially engaged in ESCC upon interaction with the NBDs. While the analyses are consistent with possible involvement of these mechanisms, they do not constitute experimental validation and should be interpreted as computational indications rather than confirmatory evidence. This study provides insights into the potential anticancer activities and the mechanistic pathway of NBDs in ESCC. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-026-00934-8.