Bile metabolite palmitic acid augments the migration of gallbladder cancer cells through the ROS/NF-кB signaling pathway.

Bile reflux, resulting from pancreaticobiliary reflux (PBR), not only alters the chemical of bile but also constitutes a significant risk factor for the occurrence and development of gallbladder cancer. In previous studies, the authors identified a marked elevation of palmitic acid (PA) levels in the bile of patients with PBR. This study seeks to elucidate the mechanisms of promoting the migration of gallbladder cancer cells, with the objective of contributing novel strategies and theoretical foundations for the treatment of gallbladder cancer. We performed a cytotoxicity screening on the NOZ and GBC-SD human gallbladder cancer cell line using varying concentrations of palmitic acid. These following methodologies were employed to investigate the mechanism of PA in NOZ and GBC-SD cells. Intracellular lipid droplet accumulation was assessed using Oil red O staining, while cell migration capability was evaluated through the Transwell migration assay. Reactive oxygen species (ROS) levels were quantified using the superoxide anion fluorescent probe, Dihydroethidium (DHE), in conjunction with a ROS detection kit. The expression levels of relevant genes and proteins were analyzed using Western blot (WB), quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) techniques. In NOZ and GBC-SD cells, it was observed that palmitic acid facilitates the accumulation of intracellular lipid droplets and diminishes cellular activity while augmenting the cells' migratory capacity. Furthermore, elevated concentrations of PA have been shown to increase ROS levels in NOZ and GBC-SD cells. This elevation also activates the Nuclear factor-kappa B (NF-κB) and the Nuclear factor erythroid 2-related factor 2 (NRF2)/Antioxidant Response Element (ARE) signaling pathways. The addition of the ROS inhibitor N-acetylcysteine (NAC) to NOZ and GBC-SD cells treated with high concentrations of PA effectively inhibits the enhancement of cell migration and epithelial-mesenchymal transition (EMT) induced by PA. PA promotes EMT in human gallbladder cancer cells by overproducing ROS and activating the NF-κB and NRF2/ARE signaling pathways, thereby facilitating increased migration.