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Abstract
Extracts from certain Mexican Indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sequiterpene lactones (SLs), which specifically inhibit the transcription factor NF-kappa B (Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS Lett. 402, 85-90). Here we show that SLs prevented the activation of NF-kappa B by different stimuli such as phorbol esters, tumor necrosis factor-alpha, ligation of the T-cell receptor, and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of I kappa B-alpha and I kappa B-beta by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-kappa B. SLs did neither interfere with DNA binding activity of activated NF-kappa B nor with the activity of the protein tyrosine kinases p59fyn and p60arc. Micromolar amounts of SLs prevented the induced expression of the NF-kappa B target gene intracellular adhesion molecule 1. Inhibition of NF-kappa B by SLs resulted in an enhanced cell killing of murine fibroblast cells by tumor necrosis factor-alpha. SLs lacking an exomethylene group in conjugation with the lactone function displayed no inhibitory activity on NF-kappa B. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or indirect anti-oxidant properties.
Highlights
The transcription factor NF-B is one of the key regulators of genes involved in the immune and inflammatory response
The target genes whose transcription is mainly regulated by NF-B include many cytokines, cell adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1, as well as acute-phase proteins and immunoreceptors
After 1 h of preincubation cells were stimulated for 20 min with murine TNF-␣ and total extracts were tested for DNA binding activity by Electrophoretic Mobility Shift Assay (EMSA)
Summary
Cell Culture—Jurkat T leukemia cells (subclone JR “Wurzburg”) were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, and 1% (w/v) penicillin/streptomycin (all purchased from Life Technologies, Inc., Grand Island, NY). Prior to the incubation with the ␣-IB-␣ and IB- antibodies (Santa Cruz Inc.), the membrane was blocked with 5% non-fat dry milk powder in TBST buffer (25 mM Tris/HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.1 mM MgCl2, 0.05% (v/v) Tween 20). Serial concentrations of parthenolide and isohelenin were preincubated at 30 °C with p60src or p59fyn protein in 50 l kinase buffer (30 mM Hepes, pH 7.2, 5 mM MgCl2, 5 mM MnCl2, 1 M ATP, 10 Ci of [␥-32P]ATP) containing 5 g of acid-treated rabbit muscle enolase (Sigma). At the indicated times 20 l of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (5 mg/ml phosphate-buffered saline) was added to all wells After another 3-h incubation, supernatants were removed followed by addition of 100 l of a 24:1 (v/v) isopropyl alcohol/ HCl solution. Dead cells were excluded by forward/side scatter gating and staining with propidium iodide
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