Cytotoxic, apoptotic and genotoxic effects of thymoquinone‑oxime derivative on gastric cancer cells: An <i>in vitro</i> study

Gastric cancer (GC) remains a notable global health concern, emphasizing the need for novel and effective therapeutic agents. The present study investigated the cytotoxic, genotoxic and apoptotic effects of thymoquinone-oxime (TQ-ox) on human gastric adenocarcinoma AGS cells and assessed its potential to induce DNA damage. Cytotoxicity was evaluated in AGS and normal human gastric epithelial cells (HGEpiCs) using a luminometric ATP assay. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured fluorometrically, whereas intracellular glutathione (GSH) content was determined via a luminometric GSH assay kit. DNA damage was quantified by a comet assay, and apoptosis was assessed by fluorescence microscopy with acridine orange/ethidium bromide (AO/EB) double staining. TQ-ox exhibited dose-dependent cytotoxicity in gastric cells, with AGS cells having a slightly higher sensitivity to TQ-ox treatment than HGEpiCs [half-maximal inhibitory concentration (IC₅₀): 40.29 vs. 46.42 µM]. Treatment with TQ-ox significantly increased intracellular ROS levels and DNA damage, while also inducing a significant depletion of intracellular GSH and a reduction of MMP, indicating an increase in oxidative stress (OS) and mitochondrial dysfunction. AO/EB staining supported a dose-dependent increase in apoptosis of gastric cells at sub-IC₅₀ concentrations of TQ-ox. Similarly, comet assay results revealed greater genotoxic effects in AGS cells compared with HGEpiCs, particularly at higher doses of TQ-ox. These findings demonstrated that TQ-ox exerted cytotoxic, pro-apoptotic and genotoxic effects on GC cells, likely mediated by OS, mitochondrial impairment and DNA damage. Taken together, these results provide additional evidence supporting the mechanistic effects of TQ-ox on GC cells and highlighted its potential as a candidate molecule for further preclinical evaluation.