Transcription factors of the NF-kappaB/Rel family are critical for inducible expression of multiple genes involved in inflammatory responses. Sulfasalazine and its salicylate moiety 5-aminosalicylic acid are among the most effective agents for treating inflammatory bowel disease and rheumatoid arthritis. However, the mode of action of these drugs remains unclear. Here we provide evidence that the transcription factor NF-kappaB is a target of sulfasalazine-mediated immunosuppression. Treatment of SW620 colon cells with sulfasalazine inhibited TNFalpha-, LPS-, or phorbol ester- induced NF-kappaB activation. NF-kappaB-dependent transcription was inhibited by sulfasalazine at micro- to millimolar concentrations. In contrast, 5-aminosalicylic acid or sulfapyridine did not block NF-kappaB activation at all doses tested. TNFalpha-induced nuclear translocation of NF-kappaB was prevented by sulfasalazine through inhibition of IkappaBalpha degradation. When blocking proteasome-mediated degradation of IkappaBalpha, we could demonstrate that sulfasalazine interfered with IkappaBalpha phosphorylation, suggesting a direct effect on an IkappaBalpha kinase or on an upstream signal. Inhibition of NF-kappaB activation seems to be specific since other DNA-binding activities such as AP1 were not affected. These results demonstrate that sulfasalazine is a potent and specific inhibitor of NF-kappaB activation, and thus may explain some of the known biological properties of sulfasalazine.
C Wahl, S Liptay, G Adler, R M Schmid
Submitter: Phillip P. Minghetti, Ph.D. | firstname.lastname@example.org
University of Alabama-Birmingham
Published November 5, 1998
I read the article "Sulfasalazine: a Potent and Specific Inhibitor of Nuclear Factor Kappa b" (Wahl et al. 1998. J. Clin. Invest.101:1163-1174.) with enthusiasm, but I have some reservations about the concentration of drug used to generate in vitro results. The authors report that the anti-inflammatory drug Sulfasalazine (salicyazosulfapyridine or SASP) is a "potent and specific" inhibitor of nuclear transcription factor kappa b (NF-kb) in the human colonic epithelial cell line SW 620. In particular, they showed a series of molecular biology experiments in which they reported the following: (a) SASP is a specific inhibitor of NF-kb activation (1000-5000 mM SASP was used; Fig. 4 ), (b) SASP blocks TNF-a and LPS-induced NF-kb-dependent transcription (200-5000 mM SASP was used; Fig. 5), (c) SASP significantly decreased Ikba steady-state mRNA levels (5000 mM SASP was used; Fig. 6 ) and (d) SASP inhibits nuclear translocation of RelA/p65 ( 5000 mM SASP was used; Fig 7).
The article reported impressive molecular biology data regarding the mechanism of action of SASP in human SW620 cells. However, what immediately struck me as a glaring discrepancy was the concentration of drug the authors used to generate results compared to the steady-state or median serum concentration of SASP seen in rheumatoid arthritis (RA) patients undergoing treatment with SASP. The concentration of SASP that Wahl et al. used to generate results (200-5000 mM ) is 13-333 times greater than the reported median level found in serum of patients undergoing treatment for rheumatoid arthritis (Smedegard and Bjork. 1995. Sulphasalazine: Mechanism of Action in Rheumatoid Arthritis. Brit. J. Rheum. 34(Suppl. 2):7-15; Schroeder and Campbell. 1972. Absorption, metabolism and excretion of salicyazosulfapyridine in man. Clin. Pharmacol. Ther.13:539-551). Specifically, the median serum concentration of SASP in RA patients administered an oral dose of 2 grams/day has been reported to be 10-15 mM. Clearly, the studies reported by Wahl et al. seem unrealistic compared to the median serum concentration seen in these patients. The results reported could thus be misleading in terms of the "potency and specificity," since excessively high concentrations of SASP were used, and it is the opinion of this writer that the clinical and scientific community should be made aware of this large discrepancy.
It is also the opinion of this writer that the experiments reported by Wahl et al. should be repeated at reported median serum concentrations of SASP to determine if the drug remains a "potent and specific" inhibitor of NF-kb activation, or its other reported immunosuppressive effects. If the results can be reproduced at the 15 mM level (+/- 5-10 mM) it would confirm and make the original data reported by Wahl et. al. believable in context of clinical serum levels of SASP. Such studies are worth pursuing, as SASP is an important anti-inflammatory drug currently used to treat rheumatoid arthritis as well as inflammatory bowel disease.
Of lesser concern is the fact that the authors used 10% fetal cow serum to conduct their in vitro cell culture studies. In a strictly scientific sense, it would be interesting to know if the same results could be obtained with SASP using 90-100% adult human serum instead of 10% fetal cow serum. In other words, design experiments that closely resemble or mimic the human serum clinical condition of a patient undergoing treatment with this drug. Although the majority of laboratories conducting cell culture experiments use 10% fetal bovine serum, including myself, it would be interesting to know if the results reported by Wahl et al. on inhibition of NF-kb activation by SASP could be reproduced using the above experimental changes.