Vanadate-induced cell growth regulation and the role of reactive oxygen species

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 O^2·− 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 Cdc2^Tyr15, 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.