Figure 2

Sulindac sulfide-induced pro-inflammatory gene up-regulation is dependent on NF-κB activity but is not mediated by apoptosis. (A) Colorimetric assay of p65 DNA binding activity to NF-κB response element in nuclear lysates of HCT-15 cells. Cells were treated with the control or SS for 4 hours and where indicated 10 ng/ml TNFα was added for the last 40 minutes before cell lysis. The DNA-binding activity of p65 is expressed as the optical density at 450 nm. Error bars indicate SEM. (B) qPCR analysis for A20, IL-8 and ICAM-1 mRNA expression levels. HCT-15 cells were pretreated with or without 50 μM PDTC for 1.5 hours, followed by treatment with the control (DMSO), 50 μM sulindac sulfide (SS), 10 ng/ml TNFα or both in combination for 4 hours. Bars represent fold change of relative gene expression, normalized to the house-keeping gene GAPDH. Mean values ± SEM (from 3 to 4 independent experiments). (C) Quantification of apoptosis and necrosis of cells treated with 50 μM SS, PDTC or both compounds in combination for 4 hours, assessed by FACS analysis of Annexin-V-Fluos/PI stained cells. Bars represent mean percentage of cells in each population ± SEM (n = 3). (D) HCT-15 cells were pre-treated with 20 μM pan-caspase inhibitor Q-VD-OPh or the control DMSO for 1 hour and then stimulated with 50 μM sulindac sulfide (SS) or the vehicle DMSO (control) for 2 hours A western blot analysis for cleaved caspase 3 (17/19 kDa) and the house-keeping gene β-actin. (E) qPCR analysis for A20, ICAM1 and IL-8 in cells treated with the indicated concentrations of SS in the presence or absence of Q-VD-Oph for 2 hours. The data are presented as fold change ± SEM (from 3 independent experiments).