Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function

Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Z(ras122), Z(ras220), and Z(ras521)) or Raf-1 (Z(raf322)) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Z(ras220) inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Z(raf322) inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-014-0082-3) contains supplementary material, which is available to authorized users.