Effect of GSPs and erlotinib on NF-κB activation in OSC19 cells.

<p>(<b>A</b>) Treatment of OSC19 cells with GSPs decreases the basal levels of NF-κB/p65 and IKKα while inhibiting the degradation of IκBα. After treatment of cells for 48 h with various concentrations of GSPs the cells were harvested, cytosolic and nuclear fractions were prepared and these were subjected to western blot analysis. Representative blots are shown from three independent experiments with identical observations. (<b>B</b>) The activity of NF-κB/p65 in the nuclear fraction of cells after treatment with and without GSPs was measured using an NF-κB/p65-specific activity assay kit, n = 3. Activity of NF-κB/p65 is expressed in terms of percent of the control (non-GSPs-treated) group. Significant decrease versus control: ^¶<i>P</i><0.05, ^*<i>P</i><0.001. (<b>C</b>) Similarly, treatment of OSC19 cells with erlotinib reduced the levels of NF-κB and IKKα. Representative blots are shown from two independent experiments. (<b>D</b>) The activity of NF-κB/p65 in the nuclear fraction of cells after treatment with and without erlotinib for 48 h was measured using the NF-κB/p65-specific activity assay kit, n = 2. Activity of NF-κB/p65 is expressed in terms of percent of control (non-erlotinib-treated) group. Significant decrease versus control: ^*<i>P</i><0.001. (<b>E</b>) Treatment of OSC19 cells with caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB, for 48 h inhibited cell invasion in a concentration-dependent manner. Data on cell invasion are summarized as the mean number of invasive cells±SD/microscopic field from three independent experiments, magnification: ×10. Significant inhibition <i>versus</i> non-CAPE-treated cells, ^*<i>P</i><0.001, ^¶<i>P</i><0.05. (<b>F</b>) GSPs reduce the levels of NF-κB-targeted proteins, such as MMP-2, MMP-9, COX-2, iNOS and VEGF, in OSC19 cells. After treatment of cells with and without GSPs for 48 h, cells were harvested, and cell lysates were subjected to the analysis of the levels of proteins using western blot analysis. Representative blots are shown from three independent experiments.