OBJECTIVE: Compound T-614, a member of the methanesulfoanilide class of anti-inflammatory agents, shows potent anti-arthritic activity in animal models of rheumatoid arthritis. The aim of the present investigation was to characterize the anti-arthritic activity of T-614 in terms of regulation of the nuclear transcription factor NF-kappaB, which is associated with expression of many immune and inflammatory genes. MATERIALS AND METHODS: THP-1 cells (human monocytic leukemia cell line) were used throughout this in vitro study, and lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha were employed for activation of the cells. Cytokine production was assayed by enzyme-linked immunosorbent assay (ELISA). The mRNA levels were determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Assessment of the NF-kappaB DNA binding activity was performed by an electrophoretic mobility shift assay (EMSA) using a digoxigenin (DIG)-labeled double-stranded oligonucleotide containing kappaB-binding site. Degradation kinetics of the cytosolic NF-kappaB inhibitor a (IkappaBalpha) were studied by Western blot analysis. RESULTS: T-614 inhibited LPS-stimulated production of TNF-alpha, interleukin (IL)-6, and IL-8 in a concentration-dependent manner with decreasing mRNA levels (IL-6 and IL-8). EMSA study showed that T-614 prevented TNF-alpha as well as LPS-stimulated activation of NF-kappaB, and Western blot analysis proved that T-614 did not affect degradation of IkappaBalpha protein. CONCLUSIONS: These results suggest that the inhibitory effect of T-614 on the production of TNF-alpha, IL-6 and IL-8 in LPS-stimulated THP-1 cells may involve transcriptional regulation through suppression of NF-kappaB activation without interfering with IkappaBalpha degradation.
OBJECTIVE: Compound T-614, a member of the methanesulfoanilide class of anti-inflammatory agents, shows potent anti-arthritic activity in animal models of rheumatoid arthritis. The aim of the present investigation was to characterize the anti-arthritic activity of T-614 in terms of regulation of the nuclear transcription factor NF-kappaB, which is associated with expression of many immune and inflammatory genes. MATERIALS AND METHODS: THP-1 cells (human monocytic leukemia cell line) were used throughout this in vitro study, and lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha were employed for activation of the cells. Cytokine production was assayed by enzyme-linked immunosorbent assay (ELISA). The mRNA levels were determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Assessment of the NF-kappaB DNA binding activity was performed by an electrophoretic mobility shift assay (EMSA) using a digoxigenin (DIG)-labeled double-stranded oligonucleotide containing kappaB-binding site. Degradation kinetics of the cytosolic NF-kappaB inhibitor a (IkappaBalpha) were studied by Western blot analysis. RESULTS:T-614 inhibited LPS-stimulated production of TNF-alpha, interleukin (IL)-6, and IL-8 in a concentration-dependent manner with decreasing mRNA levels (IL-6 and IL-8). EMSA study showed that T-614 prevented TNF-alpha as well as LPS-stimulated activation of NF-kappaB, and Western blot analysis proved that T-614 did not affect degradation of IkappaBalpha protein. CONCLUSIONS: These results suggest that the inhibitory effect of T-614 on the production of TNF-alpha, IL-6 and IL-8 in LPS-stimulated THP-1 cells may involve transcriptional regulation through suppression of NF-kappaB activation without interfering with IkappaBalpha degradation.