4-trifluoromethyl derivatives of salicylate, triflusal and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid, are potent inhibitors of nuclear factor kappaB activation

Abstract

1. The effect of two derivatives of salicylate, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) and 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal), on the activation of NF-kappaB elicited by tumour necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVEC) was tested. 2. The expression of the mRNA of vascular cell adhesion molecule-1 (VCAM-1) was studied as an example of a gene the expression of which is regulated by NF-kappaB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied. 3. Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF-kappaB. The calculation of the IC50 values showed approximately 2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate. 4. Comparison of the potency of these compounds on VCAM-1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36-43% inhibition at the same concentration. 5. Inhibition of NF-kappaB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the antibody molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose-dependent inhibition of nitrite production by the L-arginine pathway via iNOS. IC50 values for this effect were 1.13+/-0.12 mM (triflusal), 1.84+/-0.34 (HTB), 6.08+/-1.53 mM (aspirin) and 9.16+/-1.9 mM (salicylate). 6. These data indicate that the incorporation of a 4-trifluoromethyl group to the salicylate molecule strongly enhances its inhibitory effect on NF-kappaB activation, VCAM-1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo-oxygenase.

Figure 1

Chemical structures of salicylic acid, aspirin, triflusal and its main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB).

Figure 2

HUVEC were incubated in the presence of 100 u ml⁻¹ of TNF-α for the times indicated. At the end of these periods nuclear extracts were collected for the assay of κB-binding activity. A control of cells incubated for 240 min in the absence of TNF-α was also included (A). The experiment shown in (B) was carried out with nuclear extracts of cells stimulated with TNF-α for 90 min. The nuclear extract protein was incubated for 15 min at 4°C with a 1 : 40 dilution of the indicated polyclonal rabbit antibodies prior to the addition of the ³²P-labelled oligonucleotide probe. The lane marked antibody indicates an experiment where the nuclear extract was incubated with the same dilution of a rabbit anti-ovalbumin antibody. The protein-oligonucleotide complexes supershifted by the antibodies are noted by an arrow. The lanes marked competitor indicate that the nuclear extracts were incubated with the ³²P-labelled probe in the presence of a 100 fold excess of unlabelled probe.

Figure 3

Effect of different concentrations of aspirin, triflusal and HTB on the activation of NF-κB elicited by TNF-α. HUVEC were incubated with TNF-α for 90 min in the presence of the indicated additions, and at the end of this period, the nuclear extract was collected and used for the assay of κB-binding activity.

Figure 4

Effect of aspirin and HTB on the activation of NF-κB produced by TNF-α. Aspirin and HTB were added 10 min before TNF-α. Nuclear extracts were collected 90 min after addition of TNF-α. The panel represents a typical experiment of seven with identical trend. The graph shows the results expressed as mean±s.e.mean of seven blots quantitated by densitometric scanning. ^*Indicates P<0.05.

Figure 5

Effect of HTB and sodium salicylate on the activation of NF-κB elicited by TNF-α. Sodium salicylate and HTB were added 10 min before TNF-α. Nuclear extracts were collected 90 min after addition of TNF-α. The panel represents a typical experiment from eight similar experiments quantitated by densitometric scanning.

Figure 6

Effect of HTB on the activation of NF-κB produced by thrombin. HUVEC were stimulated with 1 u ml⁻¹ thrombin for 90 min prior to the collection of the nuclear extract. A typical experiment is shown in (A). (B) shows the results of the densitometric scanning of the protein/oligonucleotide complexes observed in three independent experiments with two concentrations of compounds.

Figure 7

Effect of salicylates on the expression of VCAM-1 mRNA induced by TNF-α in HUVEC. Cells were incubated with 100 u ml⁻¹ TNF-α in the presence and absence of the indicated compounds. One hour thereafter, total RNA was extracted and used for RT and PCR amplification with primers designed from the sequences of VCAM-1 and human β-actin. PCR products were separated by electrophoresis in 1.8% agarose gel and quantitated by measuring the density of ethidium bromide stained bands using the Gel Doc video gel documentation system and the Molecular Analyst software from Bio-Rad Laboratories. The molecular size of the amplification products is inferred from the electrophoretic migration of the DNA markers. A typical experiment of three similar experiments is shown in (A). The histogram shows the result of the quantitation of four independent experiments with two concentrations of compounds (B).

Figure 8

Effect of triflusal, HTB, aspirin and sodium salicylate on the activation of NF-κB elicited by insoluble IgG/ovalbumin immune complexes in rat peritoneal adherent cells. Adherent peritoneal macrophages were incubated in the presence of 100 μg ml⁻¹ IgG/ovalbumin immune complexes for 2 h in the presence or absence of the additions indicated. A characteristic experiment of three with identical trend is shown in (A). (B) shows the results of the densitometric scanning of these experiments. (C) shows a representative experiment of three carried out with aspirin and salicylate (Sal).

Figure 9

Effect of triflusal, HTB, aspirin and sodium salicylate on the production of nitrite elicited by IgG/ovalbumin complexes. The drugs were added 10 min prior to the addition of 100 μg ml⁻¹ immune complexes and NO production was assayed as nitrite after 24 h. (A) shows the effect of sodium salicylate and HTB. (B) shows the effect of aspirin and triflusal. Results are expressed as mean±s.e.mean of 7–9 experiments in duplicate.