Selective Non-Steroidal Glucocorticoid Receptor Agonists Attenuate Inflammation but Do Not Impair Intestinal Epithelial Cell Restitution In Vitro

Institute of Pharmaceutical Chemistry, Goethe University Frankfurt/Main, Campus Riedberg, Frankfurt/Main, Germany.
PLoS ONE (Impact Factor: 3.23). 01/2012; 7(1):e29756. DOI: 10.1371/journal.pone.0029756
Source: PubMed


Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile.
The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis.
Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348.
Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.

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    • "In that respect, 2-(4-acetoxyphenyl)-2-chloro-N-methylethy- lammonium chloride, named compound A (CpdA), a plant-derived precursor of phenyl aziridine, is a non-steroidal SEGRM that is under intensive investigation [15] [16] [17] [18] [19] [20]. CpdA differs from classic GCs in size and structure. "
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    • "Furthermore, elevated Hsp70 levels can negatively affect the release of the pro-inflammatory high mobility group box 1 (HMGB1) [38]. Given the mechanistic resemblance between heat shock and CpdA, with respect to their effects on cytokine gene expression, IκBα degradation and NF-κB p65 nuclear translocation (Figure 2, Figure S2) in various cell systems [39], [40], [49], [50], we were not surprised to detect that CpdA can elicit a concentration-dependent, yet transient, rise in Hsp70-coding mRNA levels and promoter activity (Figure 4, 8, Figure S4) in human A549 and PC-3 cells and murine L929sA cells. These findings could be confirmed in human MCF7 breast cancer cells (Figure S5) and skins of BALB/c mice (Figure 4C), indicating species-, cell- and tissue independence of this phenomenon. "
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