The Natural Product Hymenialdisine Inhibits Interleukin-8 Production in U937 Cells by Inhibition of Nuclear Factor-κB
Abstract
The nuclear factor-κB (NF-κB) family of transcription factors have been implicated in the inducible expression of genes involved in inflammatory and immune responses. As such, a specific inhibitor of NF-κB would be a useful therapeutic agent in a variety of inflammatory disorders. The marine natural product hymenialdisine was evaluated as an inhibitor of NF-κB in U937 cells. U937 cells were transfected with either a luciferase reporter plasmid containing the human immunodeficiency virus long terminal repeat or the interleukin-8 (IL-8) core promoter, both of which are activated by NF-κB. Hymenialdisine caused a concentration-dependent decrease in luciferase production from both reporters when the cells were stimulated with tumor necrosis factor-α, lipopolysaccharide or phorbol myristate acetate. An electrophoretic mobility shift assay confirmed its activity by inhibiting DNA binding of NF-κB. Hymenialdisine was shown to be a selective inhibitor of NF-κB in that it had no effect on the binding of other transcription factors to their DNA concensus motifs; these included activator protein-1, CCAAT/enhancer binding protein and Sp1. Functional studies showed hymenialdisine to be an inhibitor of IL-8 production and IL-8 mRNA formation in the U937 cell. Investigation into the mechanism of action of hymenialdisine showed that it was not due to inhibition of protein kinase C because the selective protein kinase C inhibitor RO 32–0432 was inactive against tumor necrosis factor-α-stimulated luciferase and IL-8 production. The compound also had no effect on IκBα or IκBβ phosphorylation and degradation. Thus, hymenialdisine is a potent inhibitor of NF-κB and IL-8 production in U937 cells.
Footnotes
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Send reprint requests to: Marie C. Chabot-Fletcher, Ph.D., SmithKline Beecham Pharmaceuticals, Department of Immunopharmacology, UW2531, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406-0939. E-mail: Marie–C–Chabot-Fletcher{at}SBPHRD.com
- Abbreviations:
- NF-κB
- nuclear factor-κB
- AP-1
- activator protein-1
- CREB
- cAMP response element binding protein
- C/EBP
- CCAAT/enhancer binding protein
- DMSO
- dimethylsulfoxide
- DTT
- dithiothreitol
- EMSA
- electrophoretic mobility shift assay
- ECL
- enhanced chemiluminescence
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HIV LTR
- human immunodeficiency virus long terminal repeat
- IL-8
- interleukin-8
- LPS
- lipopolysaccharide
- NF-IL6
- nuclear factor-interleukin 6
- PMSF
- phenylmethylsulfonyl fluoride
- PMA
- phorbol myristate acetate
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- PKC
- protein kinase C
- RLU
- relative light units
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
- TNF-α
- tumor necrosis factor-α
- RT
- reverse transcriptase
- G3PDH
- glyceraldehyde 3-phosphate dehydrogenase
- PAI
- plasminogen activator inhibitor-1
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- Received November 20, 1996.
- Accepted March 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
