Constitutive high NF-κB activity in OSCC cell lines. The gel shift was performed with nuclear extracts from both control and OSCC cells. The cells were incubated with ³²P-labeled κB oligonucleotide probe. A, there is clear binding of the probe to the nuclear extracts from OSCC9 and OSCC25 cells but not in HOK cells. B, a gel-shift assay in OSCC25 is shown. p50, and to a lesser extent p65, are present in the NF-κB complex and can be super-shifted by antibodies specific to p50 and p65. The addition of excess cold NF-κB oligonucleotide probe competes out the formation of the NF-κB complex, demonstrating the specificity of NF-κB binding. Arrowheads mark the NF-κB complex.

Global gene expression profiles in OSCC identify the dysregulation of important gene families. A, cluster analysis of genes in OSCC lines is shown. The heat maps show the differentially regulated genes and their clustering into functional groups such as cytokines, growth factors, cell cycle regulation, and transcription factors. Signal intensities for genes were averaged across all replicates of the three OSCC cell lines and HOK cells. A color scale in which red represents up-regulation and green represents down-regulation depicts the expression level of the genes. B, shown is an alteration in signal transduction pathways and gene networking in OSCC development. A schematic diagram illustrates signal transduction cascades after stimulation of IL-8 or inflammatory response genes. The IL-8 activation could promote activation of JAK/STAT pathway and the receptor tyrosine pathway, which in turn would regulate the cyclin-dependent kinase pathway. The genes in red represent oncogenes, whereas genes in green are tumor suppressors.

The mRNA expression levels of candidate genes in OSCC. Quantitative real time PCR was performed for several candidate genes in all the three OSCC cell lines to verify microarray expression data. The mRNA expression levels were compared with normal HOK cells and normalized to β-actin mRNA. The -fold changes represent the mean of triplicates and show validation of the microarray data.

The role of IL-8 up-regulation in OSCC proliferation and viability. A, shown are IL-8 levels in OSCC cell lines. ELISA was used to estimate the levels of IL-8 in culture supernatants from the various cell lines. Normal HOK cells produced very low IL-8 amounts, whereas tumor cells produced significantly higher IL-8 levels ranging from 543–2216pg/ml of protein. B, shown are photomicrographs of representative OSCC cells transfected with IL-8 siRNA. The OSCC cells were seeded at 1 × 10⁵ cells per tissue culture well and transfected with IL-8 siRNA (5 and 10 nm) or control non-target siRNA (5 nm) for 72 h. A significant change was observed in cell viability within 48 to 72 h of incubation. The concentration of transfection reagent used was 0.2 μm, and the data shown are representative of three independent experiments. C, shown is the viability of cells transfected with IL-8 siRNA. Cell viability was determined by the ability of the cells to metabolically reduce MTT to a formazan dye at 72 h after transfection with IL-8 siRNA. A decrease in cell viability after 48 h in transfected cells was noted at both 5 and 10 nm IL-8 siRNA. Significant change in cell viability in transfected cells was observed after 48–72 h at both siRNA concentrations. Statistical significance between control group and the siRNA-treated group was performed by one-way analysis of variance followed by Bonferroni's multiple comparison test (*, p < 0.05; **, p < 0.01). IL-8 levels in OSCC9 (D) and OSCC25 (E) cells transfected with IL-8 siRNA are shown. The levels of IL-8 secreted into the media from OSCC cell lines were estimated by ELISA at 6, 24, 48, and 72 h after transfection with siRNA (5 or 10 nm IL-8), and the non-target control pool was transfected with 5 nm scrambled siRNA. Significant changes in IL-8 levels were observed at 48–72 h after IL-8 knockdown. Statistical significance between control group and siRNA-treated cells was performed by one-way analysis of variance followed by Bonferroni's multiple comparison test (*, p < 0.05; **, p < 0.01).

Aspirin and Velcade reduce OSCC cell viability in a dose-dependent manner. OSCC cells were cultured in DMEM/F-12 supplemented with 10% FBS containing various doses of aspirin (A) or Velcade (B). The cells were plated at a density of 1 × 10⁵ per well with drugs varying in concentrations from 0.1 to 20 mm for aspirin and 5 to 100 nm for Velcade. The cells showed significantly reduced viability at 2 mm aspirin and 25 nm Velcade. Cell viability was measured by MTT assay at 72 h and expressed relative to control cell viability. The control HOK cells did not show any effect with aspirin treatment. Velcade is a proteasome inhibitor and affects cell viability at high concentrations. The values represent the mean of multiple experiments.