Alpha-naphthoflavone induces apoptosis through endoplasmic reticulum stress via c-Src-, ROS-, MAPKs-, and arylhydrocarbon receptor-dependent pathways in HT22 hippocampal neuronal cells

Abstract

alpha-Naphthoflavone (alpha NF) is a prototype flavone, also known as a modulator of aryl hydrocarbon receptor (AhR). In the present study, we investigated the molecular mechanisms of alpha NF-induced cytotoxic effects in HT22 mouse hippocampal neuronal cells. alpha NF induced apoptotic cell death via activation of caspase-12 and -3 and increased expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitor, salubrinal, or by CHOP siRNA transfection reduced alpha NF-induced cell death. alpha NF activated mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, and inhibition of MAPKs reduced alpha NF-induced CHOP expression and cell death. alpha NF also induced accumulation of reactive oxygen species (ROS) and an antioxidant, N-acetylcysteine, reduced alpha NF-induced MAPK phosphorylation, CHOP expression, and cell death. Furthermore, alpha NF activated c-Src kinase, and inhibition of c-Src by a kinase inhibitor, SU6656, or siRNA transfection reduced alpha NF-induced ROS accumulation, MAPK activation, CHOP expression, and cell death. Inhibition of AhR by an AhR antagonist, CH223191, and siRNA transfection of AhR and AhR nuclear translocator reduced alpha NF-induced AhR-responsive luciferase activity, CHOP expression, and cell death. Finally, we found that inhibition of c-Src and MAPKs reduced alpha NF-induced transcriptional activity of AhR. Taken together, these findings suggest that alpha NF induces apoptosis through ER stress via c-Src-, ROS-, MAPKs-, and AhR-dependent pathways in HT22 cells.