Nickel Ion Inhibits Nuclear Factor-Kappa B Activity in Human Oral Squamous Cell Carcinoma

Abstract

Background:The spontaneous IL-8 secretion observed in OSCC is partially dependent on the disregulated activity of transcription factor NF-κB. Nickel compounds are well established human carcinogens, however, little is known about the influence of nickel on the spontaneous secretion of IL-8 in oral squamous cell carcinoma (OSCC) cells. The aim of the present study was to investigate whether Ni2+ ions can influence on IL-8 secretion by OSCC.Methods and Results:The IL-8 secretion was measured by ELISA. The expression of IL-8 mRNA was examined by real-time PCR. The NF-κB activity was measured by luciferase assay. The phosphorylation status and nuclear localization of NF-κB subunits were examined by Western blotting or Transfactor kit and immunofluorescence staining, respectively. The interaction of NF-κB p50 subunit and Ni2+ ions was examined by Ni2+-column pull down assay. The site-directed mutagenesis was used to generate a series of p50 mutants. Scratch motility assay was used to monitor the cell mobility. Our results demonstrated that, on the contrary to our expectations, Ni2+ ions inhibited the spontaneous secretion of IL-8. As IL-8 reduction was observed in a transcriptional level, we performed the luciferase assay and the data indicated that Ni2+ ions reduced the NF-κB activity. Measurement of p50 subunit in the nucleus and the immunofluorescence staining revealed that the inhibitory effect of Ni2+ ions was attributed to the prevention of p50 subunit accumulation to the nucleus. By Ni2+-column pull down assay, Ni2+ ions were shown to interact directly with His cluster in the N-terminus of p50 subunit. The inhibitory effect of Ni2+ ions was reverted in the transfectant expressing the His cluster-deleted p50 mutant. Moreover, Ni2+ ions inhibited the OSCC mobility in a dose dependent fashion.Conclusions:Taken together, inhibition of NF-κB activity by Ni2+ ion might be a novel therapeutic strategy for the treatment of oral cancer. © 2013 Shionome et al.