Laxifolone A Suppresses LPS/IFN-γ-Induced NO Synthesis by Attenuating NF-κB Translocation: Role of NF-κB p105 Level

Abstract

Laxifolone A is a triterpene isolated from Euonymus laxiflorus Champ. Exposure of RAW264.7 macrophages to laxifolone A concentration-dependently suppressed lipopolysaccharide/interferon-γ (LPS/IFN-γ)-induced nitrite production (IC₅₀ = 0.37 ± 0.05 μM), inducible NO synthase (iNOS) protein, and iNOS mRNA expression. Translocation of nuclear factor-κB (NF-κB) into the nucleus with subsequent activation of iNOS gene transcription is essential in NO signaling. Western blot analysis indicated that the cytosolic NF-κB/p65 was obviously decreased after LPS/IFN-γ stimulation for 30 min and this phenomenon could be reversed by laxifolone A. Similarly, a time-related NF-κB/p65 nuclear translocation induced by LPS/IFN-γ was diminished in the presence of laxifolone A. However, laxifolone A failed to interfere in LPS/IFN-γ-evoked IκB degradation. Our results also showed that LPS/IFN-γ-stimulation resulted in the degradation of NF-κB p105, the NF-κB precursor, and laxifolone A treatment significantly counteracted this effect. Furthermore, laxifolone A itself was able to enhance NF-κB p105 protein expression. In summary, these results suggest that inhibition of NF-κB p105 degradation in cytoplasm may participate in the abrogation of LPS/IFN-γ-induced NF-κB translocation and subsequent NO synthesis by laxifolone A.

Laxifolone A - NO - INOS - NF-κB - IκB - NF-κB p105

References

• 1 Fox S B, Gatter K C, Bicknell R, Going J J, Stanton P, Cooke T G, Harris A L. Relationship of endothelial cell proliferation to tumor vascularity in human breast cancer.  Cancer Res. 1993;  53 4161-3
• 2 Verheul H M, Bom J B, Hoekman K, Pinedo H M. Inhibition of tumor angiogenesis as a cancer treatment.  Nederlands Tijdschrift voor Geneeskunde. 1999;  143 1549-55
• 3 Jenkins D C, Charles I G, Thomsen L L, Moss D W, Holmes L S, Baylis S A, Rhodes P, Westmore K, Emson P C, Moncada S. Roles of nitric oxide in tumor growth.  Proc Natl Acad Sci USA. 1995;  92 4392-6
• 4 Jenkins D C, Charles I G, Baylis S A, Lelchuk R, Radomski M W, Moncada S. Human colon cancer cell lines show a diverse pattern of nitric oxide synthase gene expression and nitric oxide generation.  Br J Cancer. 1994;  70 847-9
• 5 Perkins N D. NF-κB: tumor promoter or suppressor.  Trend Cell Biol. 2004;  14 64-9
• 6 Bharti A C, Aggarwal B B. Nuclear factor-kappa B and cancer: its role in prevention and therapy.  Biochem Pharmacol. 2002;  64 883-8
• 7 Baeuerle P A, Baltimore D. I kappa B: A specific inhibitor of the NF-kappa B transcription factor.  Science. 1988;  242 540-6
• 8 Li C C, Dai R M, Longo D L. Inactivation of NF-kappa B inhibitor I kappa Bα: Ubiquitin-dependent proteolysis and its degradation product.  Biochem Biophys Res Commun. 1995;  215 292-301
• 9 Fan C M, Maniatis T. Generation of p50 subunit of NF-kappa B by processing of p105 through ATP-dependent pathway.  Nature. 1991;  354 395-8
• 10 Liou H C, Nolan G P, Ghosh S, Fujita T, Baltimore D. The NF-kappa B p50 precursor, p105, contains an internal I kappa B-like inhibitor that preferentially inhibits p50.  EMBO J. 1992;  11 3003-9
• 11 Duan H, Takaishi Y, Imakura Y, Jia Y, Li D, Cosentino L M, Lee K H. Sesquiterpene alkaloids from Tripterygium hypoglaucum and Tripterygium wilfordii: a new class of potent anti-HIV agents.  J Nat Prod. 2000;  63 357-61
• 12 Duan H, Takaishi Y, Momota H, Ohmoto Y, Taki T, Jia Y, Li D. Immunosuppressive sesquiterpene alkaloids from Tripterygium wilfordii .  J Nat Prod. 2001;  64 582-7
• 13 Takaishi Y, Ohshima S, Nakano K, Tomimatsu T, Tokuda H, Nishino H, Iwashima A. Structures of sesquiterpene polyol esters from Celastrus stephanotiifolius with potential tumor-promotion inhibitory activity.  J Nat Prod. 1993;  56 815-24
• 14 Cheng C Y, Huang P H. Flora Reipublicae Popularis Sinicae. Beijing; Science Press 1999 Vol. 45, No. 3
• 15 Kuo Y H, Huang H C, Chiou W F, Shi L S, Wu T S, Wu Y C. A novel NO-production-inhibiting triterpene and cytotoxicity of known alkaloids from Euonymus laxiflorus .  J Nat Prod. 2003;  66 554-7
• 16 Chiou W F, Chen C F, Lin J J. Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide.  Br J Pharmacol. 2000;  129 1553-60
• 17 Hsu S M, Chen Y C, Jiang M C. 17β-Estradiol inhibits tumor necrosis factor-α-induced nuclear factor-κB activation by increasing nuclear factor-κB p105 level in MCF-7 breast cancer cells.  Biochem Biophy Res Commun. 2000;  279 47-52
• 18 Spivey A C, Weston M, Woodhead S. Celastraceae sesquiterpenoids: biological activity and synthesis.  Chem Soc Rev. 2002;  31 43-59
• 19 Gonzalez A G, Tincusi B M, Bazzocchi I L, Tokuda H, Nishino H, Konoshima T, Jimenez I A, Ravelo A G. Anti-tumor promoting effects of sesquiterpenes from Maytenus cuzcoina (Celastraceae).  Bioorg Med Chem. 2000;  8 1773-8
• 20 Thomsen L L, Miles D W. Role of nitric oxide in tumor progression: lessons from human tumours.  Cancer Metastasis Rev. 1998;  17 107-18
• 21 Ziche M. Role of nitric oxide in the angiogenesis of a vascular tissue.  Osteoarthritis Cartilage. 1999;  7 403-5
• 22 Wall M E, Wani M C. Camptothecin and taxol: from discovery to clinic.  J Ethnopharmacol. 1996;  51 239-54
• 23 Chiou W F, Chou C J, Chen C F. Camptothecin suppresses nitric oxide biosynthesis in RAW264.7 macrophages.  Life Sci. 2001;  69 625-35
• 24 Xie Q W, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase.  J Biol Chem. 1994;  269 4705-8
• 25 Thanos D, Maniatis T. NF-kappa B: a lesson in family values.  Cell. 1995;  80 529-32
• 26 Baldwin A S. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappa B.  J Clin Invest. 2001;  107 241-6
• 27 Naumann M, Scheidereit C. Activation of NF-kappa B in vivo is regulated by multiple phosphorylations.  EMBO J. 1994;  13 4597-607