Leflunomide Suppresses TNF-Induced Cellular Responses: Effects on NF-κB, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis

Abstract

Leflunomide is a pyrimidine biosynthesis inhibitor that has recently been approved for treatment of rheumatoid arthritis. However, the mechanism of leflunomide’s antiarthritis activity and is not fully understood. The critical role that TNF plays in rheumatoid arthritis led us to postulate that leflunomide blocks TNF signaling. Previously, we have demonstrated that leflunomide inhibits TNF-induced NF-κB activation by suppressing I-κBα (inhibitory subunit of NF-κB) degradation. We in this study show that leflunomide also blocks NF-κB reporter gene expression induced by TNFR1, TNFR-associated factor 2, and NF-κB-inducing kinase (NIK), but not that activated by the p65 subunit of NF-κB, suggesting that leflunomide acts downstream of NIK. Leflunomide suppressed TNF-induced phosphorylation of I-κBα, as well as activation of I-κBα kinase-β located downstream to NIK. Leflunomide also inhibited TNF-induced activation of AP-1 and the c-Jun N-terminal protein kinase activation. TNF-mediated cytotoxicity and caspase-induced poly(ADP-ribose) polymerase cleavage were also completely abrogated by treatment of Jurkat T cells with leflunomide. Leflunomide suppressed TNF-induced reactive oxygen intermediate generation and lipid peroxidation, which may explain most of its effects on TNF signaling. The suppressive effects of leflunomide on TNF signaling were completely reversible by uridine, indicating a critical role for pyrimidine biosynthesis in TNF-mediated cellular responses. Overall, our results suggest that suppression of TNF signaling is one of the possible mechanisms for inhibitory activity of leflunomide against rheumatoid arthritis.

Accelerated hepatocellular carcinoma development in mice expressing the Pim-3 transgene selectively in the liver

“…Furthermore, TNF-a can promote angiogenesis, an essential process for tumorigenesis, by inducing the production of angiogenic factors, such as vascular endothelial growth factor and hepatocyte growth factor (Tamura et al, 1993;Yoshida et al, 1997). The production of TNF-a was regulated at several steps and the first one was at the transcription level, governed by transcription factors, such as NF-kB and Activator protein-1AP-1 (Manna et al, 2000;Udalova and Kwiatkowski, 2001;Chung et al, 2007). As Pim-1 can enhance NF-kB transcriptional activity (Zemskova et al, 2008), Pim-3 might be able to similarly activate NF-kB, thereby inducing TNFa expression.…”

Geniposide inhibits high glucose-induced cell adhesion through the NF-κB signaling pathway in human umbilical vein endothelial cells

“…For example, Shimada et al reported that maximal NF-κB activity was observed at 30 min and sustained for 2 h in human umbilical vein endothelial cells (HUVECs) after treatment with lysophosphatidic acid, a proinflammatory lysophospholipid, but the expression VCAM-1 and ICAM-1 was delayed, peaking at 4 h and remaining for up to 8 h post-drug treatment [39] . In another report, Manna et al observed that NF-κB activation induced by tumor necrosis factor (TNF) can be observed 15 min after stimulation, whereas the earliest time reactive oxygen intermediate (ROI) generation was observed was 1 h poststimulation [40] . Our study also showed that geniposide had the same inhibition ratio of high glucose-induced NF-κB activation and VCAM-1 expression as the NF-κB inhibitor BAY 11-7082.…”

“…The HUVECs were seeded at a density of 5×10 3 cells/well in 96-well plates and cultured for 24 h. Then the cells were treated with or without various concentrations of geniposide (1,5,10,20,40 and 80 μg/mL). After 24 h, cell viability was measured using the Cell Counting Kit-8.…”

Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-κB–regulated antiapoptotic and cell survival gene products in human multiple myeloma cells

“…26 The whole-cell extracts were prepared by lysing resveratroltreated cells in lysis buffer (20 mM Tris [pH 7.4], 250 mM NaCl, 2 mM EDTA [pH 8.0], 0.1% Triton X-100, 0.01 mg/mL aprotinin, 0.005 mg/mL leupeptin, 0.4 mM phenylmethylsulphonylfluoride, and 4 mM sodium orthovanadate). After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with various antibodies overnight at 4°C.…”