Luteolin induces cholangiocarcinoma cell apoptosis through the mitochondrial-dependent pathway mediated by reactive oxygen species

Objectives To investigate the apoptosis-inducing effect and underlying mechanisms of luteolin in cholangiocarcinoma (CCA) cells. Methods Cell viability was determined by sulphorhodamine B. Apoptosis was detected using acridine orange/ethidium bromide dye staining and annexin V/PI staining followed by flow cytometry. The effect of luteolin on the oxidative status of CCA cells was evaluated by measuring intracellular reactive oxygen species (ROS) levels using the dihydroethidium method and quantifying glutathione levels. The mitochondria transmembrane potential (Delta Psi m) was examined through JC-1 staining. The protein levels were determined by Western blot. Caspase activity was determined using specific fluorogenic substrates. Key findings Luteolin decreased KKU-100 CCA cells' viability by induction of apoptosis. Luteolin treatment increased ROS production and decreased glutathione levels. These changes were associated with the decrease of Nrf2, gamma-glutamylcysteine ligase and heme oxygenase-1 proteins. Moreover, luteolin induced mitochondrial depolarization, which was accompanied by the release of cytochrome c and a decrease of Bcl-2 and Bcl-XL proteins. Pretreatment with antioxidants, 4-hydroxy-TEMPO and N-acetyl-L-cysteine significantly prevented luteolin-induced CCA cell death and loss of Delta Psi m. In addition, luteolin induced the activation of caspase-9 and caspase-3. Conclusions Luteolin exerts its pro-apoptotic action partly through generating intracellular ROS that then contributes to the activation of mitochondria-mediated apoptotic cell death.