Novel nitric oxide-releasing triptolidenol derivatives suppress gastric cancer by targeting the folate one-carbon metabolism pathway and inducing ROS accumulation.

Gastric cancer (GC) remains a leading cancer-related cause of death globally, with limited effective therapies due to drug resistance and heterogeneity. Triptolidenol (TPO), a diterpenoid derived from Tripterygium wilfordii Hook. f., exhibits enhanced water solubility and safety compared with triptolide (TPL), albeit with reduced antitumor activity. To address this trade-off, nitric oxide (NO)-releasing TPO derivatives were synthesized by conjugating TPO to furoxan, and compound A9 was identified as the most potent candidate. A9 exhibited sub-micromolar inhibitory activity against GC cells (HGC27 cells: IC = 0.10 ± 0.01 μM; AGS cells: IC = 0.02 ± 0.01 μM) in vitro, outperforming TPO. Mechanistically, A9 exerts synergistic antitumor effects through two distinct pathways. First, it releases NO to induce a mitochondrial ROS (Mito-ROS) burst, which in turn triggers mitochondrial dysfunction and activates the Bax/Bcl-2-Cyt c-Caspase-9/Caspase-3 apoptotic pathway. Second, A9 targets the folate one-carbon metabolism (FOCM) enzymes SHMT2 and MTHFD2, leading to decreased NADPH/NADP and GSH/GSSG ratios, which disrupts redox homeostasis and further amplifies intracellular ROS accumulation. In the HGC27 xenograft model, 15 mg/kg A9 achieved 98.78% tumor growth inhibition (TGI). Collectively, A9 integrates NO-mediated ROS induction and FOCM inhibition, emerging as a promising lead compound for the preclinical development of gastric cancer therapeutics.