Research Article

Saikosaponin-d Enhances the Anticancer Potency of TNF- via Overcoming Its Undesirable Response of Activating NF-Kappa B Signalling in Cancer Cells

Figure 2

Suppressive effect of Ssd on the TNF-α-induced NF-κB signaling. (a) Effect of Ssd on TNF-α-induced degradation of IκBα. Upper panel, TNF-α-induced the degradation of IκBα; lower panel, Ssd inhibited TNF-α induced degradation of IκBα. HepG2 cells were pretreated with indicated concentrations of Ssd for 60 min and then incubated with 20 ng/mL TNF-α at 37°C for 15 min. The IκBα in cytosolic extracts was detected by Western blotting. (b) Effect of Ssd on TNF-α-induced phosphorylation and nuclear translocation of NF-κB p65. Upper panel, Ssd inhibited TNF-α-induced nuclear translocation of NF-κB p65; lower panel, Ssd inhibited TNF-α induced phosphorylation of NF-κB p65. HepG2 cells were preincubated with indicated concentrations of Ssd for 60 min, and then the cells were treated with 20 ng/mL TNF-α for 15 min. The cytosolic and nuclear extracts were harvested for detection of p65, while the total cell extracts were harvested for the detection of phosphorylated form of p65. (c) Immunocytochemical analysis of NF-κB p65 nuclear translocation. HeLa cells were pretreated with 10 μM Ssd for 60 min, and then cells were treated with TNF-α for 30 min. Cells were fixed in 4% paraformaldehyde and costained with anti-p65 antibodies (green) and DAPI (blue). Arrows indicate the cells with p65 being translocated into nucleus under magnification 400x. (d) Cytotoxicity of Ssd with or without TNF-α in constitutively active IKK-β transfected HepG2 cells. HepG2 cells were transfected with or without c.IKK-β plasmid being subjected to Ssd treatment in the presence or absence of TNF-α (20 ng/mL) for 72 h. The cell viability was determined by MTT assay. Results are mean ± SD from three independent experiments.