title: Vanadate-induced cell growth regulation and the role of reactive oxygen species.
abstract: While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (*OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O2*-), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O2*- by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O2*- in turn generated H2O2, which reacted with vanadium(IV) to generate *OH radical through a Fenton-type reaction (V(IV) + H2O2 --> V(V) +*OH + OH-). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1 expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1 levels. Superoxide dismutase, a scavenger of O2*-, or sodium formate, an inhibitor of *OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2 is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex.
authors.affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: C
authors.initials: C
authors.lastname: Huang
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: S S
authors.initials: SS
authors.lastname: Leonard
authors.forename: R
authors.initials: R
authors.lastname: Lanciotti
authors.forename: L
authors.initials: L
authors.lastname: Butterworth
authors.forename: X
authors.initials: X
authors.lastname: Shi
mesh_terms: D000838:Anions; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002374:Catalase / Q000737:chemistry; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D000071877:Checkpoint Kinase 1; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004578:Electron Spin Resonance Spectroscopy; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008168:Lung / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase / Q000737:chemistry; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology*; D020687:cdc25 Phosphatases / Q000378:metabolism
chemical_list: D000838:Anions; D000975:Antioxidants; C534699:CCNB1 protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D017382:Reactive Oxygen Species; D013481:Superoxides; D014638:Vanadates; D009249:NADP; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D010100:Oxygen