Set-up of a cell model useful to study the effect of NF-κB inhibitors on MCPs production

F Mancini, E Castelli, C Bartella, C Apicella, I Coletta, M Vitiello, G Mangano. ACRAF S.p.A., In vitro Preclinical Development, Italy

NF-κB transcription factors have a key role in many physio-pathological processes. Aberrant regulation of NF-κB is involved in cancer development and autoimmune diseases. The NF-κB family is composed of five members: p65, RelB, cRel, p50 and p52 that form homo/heterodimeric complexes with different activities. Several distinct NF-κB activation pathways have been identified. The canonical pathway is induced by inflammatory stimuli through the IκB kinase (IKK complex). A rapid phosphorylation of IκBα followed from ubiquitin-proteasome proteolysis leads to the activation of p50/p65 and the transcription of target genes (eg. MCPs). Strategies for blocking NF-κB include upstream inhibition of NF-κB activation using proteasome inhibitors (bortezomib), IKK inhibitors (NSAIDs, sulfasalazine, curcumin and parthenolide analogs), direct competitive inhibitors such as cell-permeable peptides and interfering mRNA. Developing drugs which can control specific subsets of NF-κB targets without disrupting the whole set of inflammatory genes could be a useful therapeutic approach [1].

The aim of this study is to identify an in vitro model to study selective inhibition of the MCPs synthetic pathway targeting NF-κB subunits. Some inhibitors of NF-κB pathway able to inhibit MCPs were assayed as pharmacological tools on the mouse macrophagic cell line RAW 264.7.

The RAW 264.7 cell system was set-up testing a panel of stimuli (LPS 0.1-1-10µg/ml; TNFα 10-100ng/ml with or without IFNγ 300U/ml, TNFα 50ng/ml with IFNγ 150U/ml) in order to find the best condition to assess MCP-1, MCP-3 and MIP-2 gene expression and protein production. LPS 0.1µg/ml was chosen as the most appropriate stimulus. The following compounds were tested to see how the chemokine system was modulated by interfering with the NF-κB pathway at different levels: PF-184 and IKK-16 (IKK inhibitors), andrographolide, an irreversible antagonist of NF-κB and AP-1 activation, JSH-23, a selective blocker of p65 nuclear translocation, MG-132 proteasome inhibitor. Compounds were assayed in the range 1nM – 50µM [2].

In these experimental conditions , andrographolide and JSH-23 were significantly effective in inhibiting MCP-1 (showing at 10µM an inhibition of 79% and 50% respectively) and MCP-3 (showing at 10µM an inhibition of 83% and 53% respectively) production. Interestingly, andrographolide and JSH-23 did not affect MIP-2 synthesis. Gene expression results are in agreement with those obtained in production experiments. The effects of andrographolide and JSH-23 were evaluated on a panel of cytokine/chemokine in order to verify the selectivity of the effects. RAW 264.7 surnatants, coming from a 24h incubation with LPS/NF-κB inhibitors, were assayed by Milliplex kit for MCP-1, MIP-1α, MIP-2, KC, TNF-α, IL-1β, IL-6. Previous results were confirmed together with an inhibiting effect on MIP-1α production. Furthermore, JSH-23, but not andrographolide, at 10µM showed inhibition on IL-6 production, while TNF-α levels induced by LPS were unaffected by both compounds.

In conclusion, data obtained show that interacting with the NF-κB pathway at various levels leads to modulation of chemokine synthesis with differential effects on the several family members. This cell system may therefore be useful to dissect the NF-κB-regulated inflammatory gene system.

References

[1] V Baund et al. Nature Reviews 2009.

[2] E Mora et al. Cell Cycle 2012.