Figure 6

Effect of chemical or molecular inhibition of NF-κB on CXCL8 secretion by SW480 cells in response to CPT. A: Reporter SW480 cells were left untreated or treated with DMSO (vehicle), NF-κB inhibitor SM7368 (10 μM), CPT (1 μM), phleomycin (100 μg/ml), or combinations of CPT or phleomycin with SM7368. Relative NF-κB reporter activity was measured by luciferase reporter assay. B-C: Parental SW480 cells were treated with DMSO (vehicle), CPT, SM7368, or a combination of CPT and SM7368 as shown and at the same concentrations as in A. The expression of CXCL8 was analyzed at mRNA level by qRT-PCR (B) or by cytokine ELISA in cell culture supernatant (C). Relative expressions in comparison to that from vehicle treated cells are indicated. D: Reporter SW480 cells were transfected with control (mock) or specific siRNA against p65 (si-p65) for 24 hours, after which they were treated with DMSO (vehicle) or CPT. NF-κB reporter activity was measured 24 hours after treatment. Cells were also treated with CPT in combination with SM-7368 for comparison. E-F: Effects of Chk1/Chk2 inhibition on NF-κB activity. NF-κB reporter SW480 cells were treated with varying concentrations (0–100nM) of the Chk1/Chk2 inhibitor AZD-7762 or 0.5 μM CPT as single agents (E) or varying concentration of AZD-7762 in combination with 0.5 μM CPT (F). NF-κB activation was measured by luciferase assay. While AZD-7762 by itself did not have an effect on NF-κB activation, it significantly (student’s T-test, p = 0.0002) inhibited CPT-induced NF-κB activation at100 nM concentration.