An anti-rheumatic agent T-614 inhibits NF-kappaB activation in LPS- and TNF-alpha-stimulated THP-1 cells 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.


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.
Aikawa
Y
Y
Research Laboratories, Toyama Chemical Co., Ltd., Japan. yukihiko_aikawa@toyama-chemical.co.jp
Yamamoto
M
M
Yamamoto
T
T
Morimoto
K
K
Tanaka
K
K
D018501:Antirheumatic Agents / Q000494:pharmacology*; D001578:Benzopyrans / Q000494:pharmacology*; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D007948:Leukemia, Monocytic, Acute / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018501:Antirheumatic Agents; D001578:Benzopyrans; D016207:Cytokines; D020672:I-kappa B Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; C077313:T 614; D000072000:NF-KappaB Inhibitor alpha