The carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cells

Abstract

Background and purpose: Recent evidence indicates that carbon monoxide-releasing molecules (CO-RMs) exhibit potential anti-inflammatory properties. In the present study, we have investigated whether tricarbonyl dichloro ruthenium(II) dimer (CORM-2) can control the inflammatory response induced by cytokines in a human colonic epithelial cell line, Caco-2.

Experimental approach: Caco-2 cells were preincubated with CORM-2 for 30 minutes and then stimulated with interleukin (IL)-1beta, tumor necrosis factor-alpha and interferon-gamma for different times. Gene expression was analyzed by real-time PCR. Protein expression was investigated by Western blot and ELISA. Transcription factor activation was determined by the luciferase method.

Key results: We have shown that CORM-2 significantly decreased the mRNA expression of nitric oxide synthase-2 (NOS-2) and the production of nitrite, in Caco-2 cells stimulated with cytokines. IL-8, IL-6 and metalloproteinase-7 (MMP-7) mRNA and protein were also significantly reduced by CORM-2. Time-course and small interfering RNA studies suggest that inhibition of IL-6 plays a role in the regulation of MMP-7 expression by CORM-2. These effects of CORM-2 can be dependent on the modulation of nuclear factor-kappaB (NF-kappaB), activator protein-1, CCAT/enhancer binding protein and the phosphorylated forms of NF-kappaB inhibitory protein-alpha, c-Jun N-terminal protein kinase 1/2, p38 and extracellular signal-regulated kinase 1/2.

Conclusions and implications: CORM-2 can regulate a number of genes relevant in intestinal inflammation and cancer progression. These findings provide new insights into the anti-inflammatory properties and potential applications of this class of compounds.

Figure 1

Effects of CORM-2 on nitrite levels in supernatants (a) and NOS-2 mRNA expression (b) in Caco-2 cells stimulated with cytomix for 18 or 12 h, respectively. Results are the mean±s.e.m. of three separate experiments each consisting of four samples. ^*P<0.05; ^**P<0.01 vs control cells stimulated with cytomix (C). (−), nonstimulated control cells.

Figure 2

Effects of CORM-2 on cell viability (a) and HO-1 protein expression (b). Caco-2 cells were incubated with test compounds for 24 h in the absence or presence of cytomix (C). Viability was expressed as percentage of nonstimulated control cells (−). Results are the mean±s.e.m. of three separate experiments. CoPP, cobalt protoporphyrin IX. The immunoblot is representative of three independent experiments. ^**P<0.01 vs control cells stimulated with cytomix (C).

Figure 3

Effects of CORM-2 on IL-8 protein levels in supernatants (a) and mRNA expression (b) in Caco-2 cells stimulated with cytomix for 18 or 12 h, respectively. Results are the mean±s.e.m. of three separate experiments, each consisting of four samples. ^*P<0.05; ^**P<0.01 vs control cells stimulated with cytomix (C). (−), nonstimulated control cells.

Figure 4

Effects of CORM-2 on pro-MMP-7 protein levels in supernatants (a) and mRNA expression (b) in Caco-2 cells stimulated with cytomix for 18 or 12 h, respectively. Results are the mean± s.e.m. of three separate experiments each consisting of three samples. ^*P<0.05; ^**P<0.01 vs control cells stimulated with cytomix (C). (−), nonstimulated control cells.

Figure 5

Effect of CORM-2 on IL-6 levels in supernatants from cells stimulated with cytomix for 6 h (a), and time course of IL-6 and MMP-7 mRNA expression and effect of CORM-2 (100 μM) (b). Results are the mean±s.e.m. of three separate experiments each consisting of four samples. (−), nonstimulated control cells. ^*P<0.05; ^**P<0.01 vs control cells stimulated with cytomix (C).

Figure 6

Influence of IL-6 modulation on the inhibitory effect of CORM-2 in MMP-7 mRNA expression in Caco-2 cells stimulated with cytomix for 12 h (a). Concentrations used were: CORM-2 (100 μM), IL-6 (1 ng ml⁻¹) and IL-6 or nonspecific siRNA (100 nM). Stimulation of MMP-7 mRNA expression by IL-6 (0.2 or 1 ng ml⁻¹) addition to Caco-2 cells and incubation for 12 h. Effect of CORM-2 (100 μM) (b). Results are the mean±s.e.m. of two separate experiments each consisting of three samples. ^**P<0.01 vs control cells stimulated with cytomix (C).

Figure 7

Effect of CORM-2 on NF-κB promoter activation (a) and NF-κB-DNA binding (b), in Caco-2 cells stimulated with cytomix for 8 h or 30 min, respectively. Results are the mean±s.e.m. of three separate experiments each consisting of three samples. (−), nonstimulated control cells. ^**P<0.01 vs control cells stimulated with cytomix (C). In (b), the figure is representative of three experiments with consistent results.

Figure 8

Effect of CORM-2 on IκBα expression (a) and IκBα phosphorylation (b), in cytoplasmic extracts from Caco-2 cells stimulated with cytomix for either 15 or 30 min. Results are the mean±s.e.m. of three separate experiments each consisting of three samples. (−), nonstimulated control cells. ^**P<0.01 vs control cells stimulated with cytomix (C). In (a), the figure is representative of three experiments with consistent results.

Figure 9

Effect of CORM-2 on AP-1 (a) and C/EBP (b) promoter activation in Caco-2 cells stimulated with cytomix for 8 h. Results are the mean±s.e.m. of two separate experiments each consisting of three samples. (−), nonstimulated control cells. ^**P<0.01 vs control cells stimulated with cytomix (C).

Figure 10

Effect of CORM-2 on the expression of the phosphorylated forms of JNK1/2, p38 and ERK1/2 in Caco-2 cells stimulated with cytomix for 15 min. (−), nonstimulated control cells. The figure is representative of three experiments with consistent results.