Non-Steroidal Anti-Inflammatory Drugs Alter Nuclear Factor- Κβ in a Class Specific Manner

Myers MJ et al. Treatment Dev Vet Pract 2020(1): 11-18. 10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice OCIMUM Research Article Non-Steroidal Anti-Inflammatory Drugs Alter Nuclear Factor- κβ in A Class Specific Manner Abstract The capacity of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) to affect the NF-κβ dependent pathway was examined. The NSAIDs were co-cultured with RAW 264.7 cells transfected with a plasmid encoding Secreted Embryonic Alkaline Phosphatase (SEAP) under the control of NF-κβ. SEAP production was initiated by stimulating the cells with E. coli derived Lipopolysaccharide Michael J Myers*, Anne J Lewandowski and (LPS). Diclofenac, indomethacin, celecoxib, tolfenamic acid, flufenamic acid, Christine M Deaver and piroxicam inhibited SEAP production. Ketoprofen, phenylbutazone, Center for Veterinary Medicine, Office of Research aspirin, and acetaminophen did not affect SEAP production. TNFα stimulation Division of Applied Veterinary Research, USA of HEK293 cells transfected with a SEAP reporter gene under the control of NF-κβ was used to determine whether the NSAIDs were working directly on Received: 16 June 2020 the NF-κβ pathway or on TLR4 signaling. TNFα stimulated HEK293 cells Accepted: 20 July 2020 showed that diclofenac and flunixin inhibited SEAP production, but aspirin Version of Record Online: 31 July 2020 did not. Meloxicam and diclofenac both inhibited the amount of p65 released after LPS stimulation of the RAW 264.7 cells while flunixin did not affect Citation p65 release. Flunixin had no effect on an NF-κβ gel shift assay. These results demonstrate that NSAIDs in four of the six NSAID families can inhibit NF- Myers MJ, Lewandowski AJ, Deaver CM (2020) κβ pathway, albeit via different points in the pathway. Non-Steroidal Anti-Inflammatory Drugs Alter Nuclear Factor- Κβ in A Class Specific Manner. Keywords Treatment Dev Vet Pract 2020(1): 11-18. Flunixin Meglumine; Gel Shift; Lipopolysaccharide; Non-Steroidal Anti- Correspondence should be addressed to Inflammatory Drugs; Nuclear Factor-Κβ Michael J Myers, USA E-mail: [email protected] Abbreviations Copyright NSAID : Non-Steroidal Anti-Inflammatory Drugs Copyright © 2020 Myers MJ et al. This is an open LPS : Lipopolysaccharide access article distributed under the Creative NF-κβ : Nuclear Factor-κβ Commons Attribution License which permits unrestricted use, distribution, and reproduction Introduction in any medium, provided the original author and work is properly cited. The most widely studied mechanism of action of Nonsteroidal Anti- Inflammatory Drugs (NSAIDs) is their reduction of eicosanoid production through the inhibition of the enzymatic activity of the Cyclooxygenase (COX) enzymes, COX1 & COX2. As a drug class, NSAIDS can exhibit antipyretic, anti-inflammatory, and/or analgesic effects; all NSAIDs do not exhibit all three activities. COX1 is constitutively expressed, while COX2 is inducible during periods of inflammation and is the more important Ocimum Scientific Publishers .01. Submit Manuscript 10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice form during inflammation. While inhibition of Prostaglandin [23] or to inhibit NF-κβ [17]. Carprofen decreases NF-κβ E2 (PGE2) production is perhaps the most widely studied activation while flunixin meglumine has no effect on -κβNF aspect of NSAIDS, other studies have shown some NSAIDS, activation [24]. including aspirin, indomethacin, diclofenac, meloxicam, tolfenamic acid, celecoxib, and flufenamic acid can inhibit The disparate reports on the impact of various NSAIDs on NF-κβ activation [1-17]. NF-κβ activation raises the question of whether the effect of NF-κβ activation is a selective effect of the different drugs, a Previous in vivo studies from this laboratory identified three concentration dependent effect, or a cell-type specific effect. swine genomic biomarkers of inflammation that held promise of Therefore, to address this issue, we used several different clinical utility [18]. More intriguing was the finding that there cell lines, each of which had different cell surface signaling in vivo expression patterns were altered by administration of pathways with the read-out system for all being the expression the Nonsteroidal Anti-Inflammatory Drug (NSAID) flunixin of Secreted Embryonic Alkaline Phosphatase (SEAP). The meglumine [19], further suggesting these biomarkers held capacity of NSAIDs in the 5 major families to affect NF-κβ the potential to be used as surrogate endpoints to assess dependent signaling, and the mechanisms of action responsible the effectiveness of novel NSAIDs with claims to control for affecting this pathway were examined, with the expectation inflammation. Critical evaluation of these results however that the pharmacological results obtained from these cell lines raised the question of whether we were developing a model would guide future in vivo studies in swine. applicable to all NSAIDs, or if we were simply developing a flunixin meglumine specific model. The central question Materials and Methods then became how to determine if these results were more broadly applicable to other NSAIDs, which would then imply Chemicals and NSAIDs a common mechanism of action underlying their capacity to Flunixin meglumine, diclofenac, meloxicam, phenylbutazone, affect gene expression. Alterations in NF-κβ activation would ketoprofen, celecoxib, indomethacin, flufenamic acid, provide both an explanation for why flunixin meglumine piroxicam, tolfenamic acid, acetomenaphen, and aspirin were suppressed in vivo gene expression Peters et al., [19] and a all obtained from Sigma-Aldrich Chemical Co (St. Louis, MO). common mechanism of action by with other class members All drugs were >98% pure. Recombinant human TNFα was would be expected to exert similar actions in vivo. obtained from Invitrogen, Inc. (Carlsbad, CA). E. coli derived NF-κβ is an essential intracellular transcription factor with a lipopolysaccharide (LPS, E. coli 055: B5) was obtained from central role in the regulation of innate and adaptive immune Sigma-Aldrich. responses, including regulation of multiple genes induced during inflammatory responses [20]. As such, NF-KB has been Cell culture postulated as a potential target for therapeutic intervention. The impact of various NSAIDs was determined using RAW However, the ability of NSAIDs to inhibit NF-κβ activation 264.7 cells transfected with a plasmid that produces SEAP has not been a constant observation with all drugs in this class. under the transcriptional control of five NF-κβ response Neither have there been consistent observations demonstrating elements (RAW 264.7-SEAP; Novus Biologicals, Littleton, CO; the same effect on NF-κβ activation by the same NSAID. Cat # NBP2-26261). The cells were grown using Ultraculture Indomethacin has been reported to either inhibit Shen et media supplemented with 4 mM L-glutamine, 1mM Na al., [6] or not inhibit NF-κβ activation. Acetaminophen, an pyruvate, 100 units/ml penicillin & 100 µg/ml streptomycin analgesic and antipyretic NSAID, was shown in one report (all from Lonza, Walkersville MD). Media for transfected cells to directly inhibit NF-κβ activation, while this same report was supplemented with 500 µg/ml G418 (Sigma-Aldrich) found aspirin only indirectly inhibited NF-κβ activation [21]. to maintain the plasmid. The cells were passaged when they Aspirin, indomethacin, diclofenac, meloxicam, tolfenamic reached 70-80% confluency using trypsin/EDTA. The impact acid, and flufenamic acid are NSAIDS with antipyretic, anti- of NSAIDs on NF-κβ was also assessed using HEK293 cells inflammatory, and analgesic effects. Tolfenamic acid has been transfected with a SEAP reporter gene under the control of an reported to induce (rather than suppress) NF-κβ activation in IFN-β minimal promoter fused to five NF-κβ (and five AP-1) colorectal cancer cells through enhanced phosphorylation and binding sites (HEK293-TNF; Invivogen, San Diego, CA; Cat degradation of Iκβ-α Jeong et al., [22], while another study # hkb-tnfil1). These cells were grown using Ultraculture media found it suppressed inflammatory induced NF-κβ activation supplemented with 4 mM L-glutamine, 1mM Na pyruvate, by decreasing the degradation of Iκβ-α [12]. Flufenamic acid 100 units/ml penicillin & 100 µg/ml streptomycin (all from has been reported to have no impact on NF-κβ activation Lonza, Walkersville MD) and Zeocin (100 µg/ml). The cells Submit Manuscript .02. Ocimum Scientific Publishers Treatments and Developments: Veterinary Practice 10.33513/TDVP/2001-02 were passaged when they reached 70-80% confluence using Gels were run and transferred in Bio-Rad equipment (Trans- trypsin/EDTA. Blot® Cell and Criterion™ Cell). Determination of SEAP production Chemiluminescence was read on a Typhoon™ 8610 Variable Mode Imager. Densitometry was performed and images were Test cells were plated into a 96-well plate at 5x104 cells/well colorized in ImageQuant™ software. (200 µl final volume) overnight to permit cell attachment. The media was replaced and the test NSAID (or control) was Data transformation and statistics added to the cells 1 hr prior to addition of the stimulating agent. NSAIDs were added to achieve final concentrations of SEAP assay results from each experiment were expressed 10-3 M to 10-7 M. RAW 264.7-SEAP cells were stimulated as percent of LPS stimulated values for each experiment, with LPS (1 µg/ml, final concentration). The HEK293-TNF with the LPS stimulated SEAP concentrations being 100%. cells were stimulated

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