title: Areca nut extract induces pyknotic necrosis in serum-starved oral cells via increasing reactive oxygen species and inhibiting GSK3β: an implication for cytopathic effects in betel quid chewers.
abstract: Areca nut has been proven to be correlated with various pathologic alterations in oral cavity. However, the mechanisms for such cytopathic effects are still elusive due mostly to the limitations of cell culture systems. Here we discovered that areca nut extract (ANE) induced production of autophagosome vacuoles in cells cultured with rich medium but induced pyknosis and ballooning, two morphological alterations frequently observed in betel quid chewers, in cells under a serum-free culture condition. Permeability of the serum-starved cells to propidium iodide (PI) confirmed ANE induced novel necrosis with pyknosis (pyknotic necrosis), providing a possible explanation for inflammatory infiltration in chewers' mucosa. In these serum-starved cells, ANE strongly induced reactive oxygen species (ROS), which acted as a key switch for the initiation of pyknotic necrosis. Calcium flux was also involved in the morphological alterations. Besides, inhibition of GSK3β by SB216763 significantly exacerbated the pyknotic necrosis either induced by ANE or H2O2 in serum-starved cells, suggesting that GSK3β is a critical regulator for ANE/ROS-mediated pyknotic necrosis. Interestingly, LC3-II transition and PARP cleavage were still detected in the serum-starved cells after ANE treatment, suggesting concurrent activation of apoptotic and autophagic pathways. Finally, insulin could counteract the effect of ANE-induced pyknotic necrosis. Taken together, these data provide a platform for studying ANE-induced cytopathogenesis and the first clinical implication for several pathological alterations, such as ballooning and inflammatory infiltration, in betel quid chewers.
authors.affiliation: Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan.
authors.forename: Wen-Tsai
authors.initials: WT
authors.lastname: Ji
authors.forename: Cheng-I
authors.initials: CI
authors.lastname: Lee
authors.forename: Jeff Yi-Fu
authors.initials: JY
authors.lastname: Chen
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Cheng
authors.forename: Sheng-Ru
authors.initials: SR
authors.lastname: Yang
authors.forename: Jung-Hua
authors.initials: JH
authors.lastname: Chen
authors.forename: Hau-Ren
authors.initials: HR
authors.lastname: Chen
mesh_terms: D001114:Areca / Q000737:chemistry*; D001343:Autophagy / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008409:Mastication / Q000187:drug effects*; D008954:Models, Biological; D009055:Mouth / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D009336:Necrosis; D010936:Plant Extracts / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D016895:Culture Media, Serum-Free; D007328:Insulin; D010936:Plant Extracts; D017382:Reactive Oxygen Species; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D020169:Caspases